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J Physiol 594.20 (2016) pp 6009–6024

Phrenic motor outputs in response to bronchopulmonary C-fibre activation following chronic cervical spinal cord injury Kun-Ze Lee1,2,3,4,5 1

Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan Centre for Neuroscience, National Sun Yat-sen University, Kaohsiung, Taiwan 3 Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Taiwan 4 Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung, Taiwan 5 Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan 2

The Journal of Physiology

Key points

r Activation of bronchopulmonary C-fibres, the main chemosensitive afferents in the lung, can induce pulmonary chemoreflexes to modulate respiratory activity.

r Following chronic cervical spinal cord injury, bronchopulmonary C-fibre activation-induced inhibition of phrenic activity was exaggerated.

r Supersensitivity of phrenic motor outputs to the inhibitory effect of bronchopulmonary r r

C-fibre activation is due to a shift of phrenic motoneuron types and slow recovery of phrenic motoneuron discharge in cervical spinal cord-injured animals. These data suggest that activation of bronchopulmonary C-fibres may retard phrenic output recovery following cervical spinal cord injury. The alteration of phenotype and discharge pattern of phrenic motoneuron enables us to understand the impact of spinal cord injury on spinal respiratory activity.

Abstract Cervical spinal injury interrupts bulbospinal pathways and results in cessation of phrenic bursting ipsilateral to the lesion. The ipsilateral phrenic activity can partially recover over weeks to months following injury due to the activation of latent crossed spinal pathways and exhibits a greater capacity to increase activity during respiratory challenges than the contralateral phrenic nerve. However, whether the bilateral phrenic nerves demonstrate differential responses to respiratory inhibitory inputs is unclear. Accordingly, the present study examined bilateral phrenic bursting in response to capsaicin-induced pulmonary chemoreflexes, a robust respiratory inhibitory stimulus. Bilateral phrenic nerve activity was recorded in anaesthetized and mechanically ventilated adult rats at 8–9 weeks after C2 hemisection (C2Hx) or C2 laminectomy. Intra-jugular capsaicin (1.5 µg kg−1 ) injection was performed to activate the bronchopulmonary C-fibres to evoke pulmonary chemoreflexes. The present results indicate that capsaicin-induced prolongation of expiratory duration was significantly attenuated in C2Hx animals. However, ipsilateral phrenic activity was robustly reduced after capsaicin treatment compared to uninjured animals. Single phrenic fibre recording experiments demonstrated that C2Hx animals had a higher proportion of late-inspiratory phrenic motoneurons that were relatively sensitive to capsaicin treatment compared to early-inspiratory phrenic motoneurons. Moreover, late-inspiratory phrenic motoneurons in C2Hx animals had a weaker discharge frequency and slower recovery time than uninjured animals. These results suggest bilateral phrenic nerves differentially respond to bronchopulmonary C-fibre activation following unilateral cervical hemisection, and the severe inhibition of phrenic bursting is due to a shift in the discharge pattern of phrenic motoneurons.

 C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

DOI: 10.1113/JP272287

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(Resubmitted 15 February 2016; accepted after revision 19 April 2016; first published online 23 April 2016) Corresponding author K.-Z. Lee: Department of Biological Sciences, College of Science, National Sun Yat-sen University, #70 Lien-Hai Rd, Kaohsiung city 804, Taiwan. Email: [email protected] Abbreviations BL, baseline; C2Hx, C2 spinal hemisection; Early-I, early-inspiratory; Late-I, late-inspiratory; P ETCO2 , partial pressure of end-tidal CO2 ; TE , expiratory duration; TI , inspiratory duration.

Introduction Phrenic motoneurons are primarily located within the ventral horn of the mid-cervical spinal cord (e.g. C4–C6 in the cat; C3–C6 in the rat and mouse) and receive inspiratory drives from the premotor neurons in the brainstem (Ellenberger & Feldman, 1988; Lane, 2011; Buttry & Goshgarian, 2014). Bilateral phrenic nerves display identical inspiratory rhythmic activity under normal conditions; however, lateral cervical spinal cord injury usually interrupts the bulbospinal respiratory pathway and causes cessation of phrenic bursting or diaphragm EMG activity ipsilateral to the lesion (Zhou et al. 2001; Tsai & Lee, 2014; Hsu & Lee, 2015; Navarrete-Opazo et al. 2015). Although inspiratory activity can partially recover over weeks to months after lateral cervical spinal cord injury (Fuller et al. 2008; Goshgarian, 2009; Lee et al. 2014; Warren et al. 2014), phrenic activity ipsilateral to the lesion is differentially regulated compared to the contralateral phrenic nerve in response to several respiratory-related stimuli. For example, Lee et al. (2010) observed that bilateral cervical vagotomy significantly enhanced the ipsilateral phrenic burst amplitude compared to the contralateral phrenic nerve at 2 and 8 weeks after unilateral hemisection at the 2nd cervical spinal cord (i.e. C2Hx). Short-term hypoxia also induced a greater enhancement in the ipsilateral phrenic burst amplitude during the chronic injury phase (Lee et al. 2015). Similarly, the phrenic response to hypercapnia was greater in the ipsilateral compared to the contralateral phrenic nerve (Fuller et al. 2006). In addition, hypoxia-induced short-term and long-term plasticity is differentially expressed between the injured and uninjured side phrenic nerve. Specifically, the capability of increasing burst amplitude following hypoxia is augmented in the ipsilateral phrenic nerve (Doperalski & Fuller, 2006; Lee et al. 2015). These data suggest that the excitability of phrenic motoneurons may be altered after cervical spinal cord injury, and increases in respiratory drives preferentially enhance phrenic motor outputs ipsilateral to a lateral cervical spinal cord injury. However, no studies have investigated whether the bilateral phrenic nerves have differential responses to inhibitory respiratory inputs. Activation of bronchopulmonary C-fibres, the main chemosensitive afferents in the lung, can suppress both supraspinal and spinal respiratory activity (Lee & Pisarri, 2001; Lin et al. 2015). Thus, the present study first examined the bilateral phrenic activity in response to

bronchopulmonary C-fibre activation following chronic cervical spinal cord injury. The phrenic nucleus is heterogeneous and composed of different types of phrenic motoneurons in humans and animals (Lee & Fuller, 2011; Butler et al. 2014). In the rat model, phrenic motoneurons are divided into early-inspiratory (Early-I), late-inspiratory (Late-I) and silent motoneurons based on the firing pattern and relative discharge onset to the whole phrenic bursting (Lee & Fuller, 2011). Several reports have demonstrated that distinct phrenic motoneuron types have divergent responses to respiratory-related stimuli. For example, high respiratory drives (e.g. hypercapnia and hypoxia) specifically induced an earlier discharge in Late-I but not Early-I phrenic motoneurons (Kong & Berger, 1986; Prabhakar et al. 1986; Lee et al. 2009). Moreover, only Late-I but not Early-I phrenic motoneurons exhibited an augmented discharge frequency during gasping (St John & Bartlett, 1981). These results suggest that Early-I and Late-I phrenic motoneurons are differentially regulated by the respiratory neural circuit. The first part of this study demonstrated that bronchopulmonary C-fibre activation significantly reduced phrenic activity ipsilateral to the lesion. We hypothesized that the altered response of the whole phrenic motor output could be due to changes in the proportion and/or discharge pattern of individual phrenic motoneuron types in chronic C2Hx animals. Accordingly, our second aim explored the motoneuron mechanisms underlying the alteration of phrenic motor output in response to bronchopulmonary C-fibre activation in uninjured and C2Hx animals. Methods Ethical approval

All experimental procedures were approved by the Institutional Animal Care and Use Committee at the National Sun Yat-sen University and were performed in accordance with the University’s guidelines for experimental animals. Animals

A total of 35 male, adult Sprague-Dawley rats (7–8 weeks of age) obtained from BioLasco Taiwan were housed in the animal room with free access to food and water. Animals

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were assigned to either the uninjured group (i.e. C2 laminectomy only) (n = 17) or the C2Hx group (n = 18). Spinal cord injury

At 8–9 weeks of age, the animals were anaesthetized via an injection of xylazine (10 mg kg−1 , S.C., Rompun, Bayer, Leverkusen, Germany) and ketamine (140 mg kg−1 , i.p., Ketalar, Pfizer, New York, USA). After an absence of the toe-pitch withdrawal reflex, the dorsal spinal cord was exposed at the C1–C3 level followed by a C2 laminectomy in all animals. A left C2 hemisection was then performed with a micro-scalpel and aspiration in C2Hx group animals. The dura was sutured with 10-0 nylon (UNIK) sutures, and the overlying muscle and skin were closed with 4-0 chromic (UNIK) and 4-0 nylon sutures (UNIK), respectively. Following the surgical procedures, yohimbine (1.2 mg kg−1 , S.C., Tocris, Ellisville, MO, USA) and lactated Ringer solution (5 ml, S.C., Nang Kuang Pharmaceutical Co., Ltd, Taiwan) were administered to reverse the effects of xylazine and prevent dehydration, respectively. An analgesic (buprenorphine, 0.03 mg kg−1 , S.C., Shinlin Sinseng Pharmaceutical Co., Ltd, Taiwan) was administered to all animals for analgesia. Post-surgical care protocols, including daily oral delivery of Nutri-Cal (1–3 ml, EVSCO Pharmaceuticals) and injection of lactated Ringer solution (5 ml, S.C.), were performed until adequate volitional drinking and eating resumed. Phrenic motor output measurement

At 8 weeks following injury, the animals (uninjured, weight: 561 ± 13 g; C2Hx, weight: 527 ± 13 g) were anaesthetized via an intraperitoneal injection of urethane (1.6 g kg−1 , Sigma, St Louis, MO, USA) and placed in a supine position. The rats’ rectal temperature was recorded by an electrical thermometer and maintained at 37 ± 1°C by a servo-controlled heating pad (model TC-1000, CWE Inc., Ardmore, PA, USA). After confirmation of adequate anaesthesia, a tracheotomy was conducted, and an endotracheal tube (PE-240, Clay Adams) was inserted into the trachea below the larynx. The right jugular vein and left femoral artery were catheterized for drug administration and blood pressure measurement (transducer: DTX-1; amplifier: TA-100, CWE Inc.), respectively. Animals were then mechanically ventilated (KDS 35, KD Scientific, Holliston, MA, USA) with a gas mixture (50 % O2 , 50 % N2 ; volume = 7 ml kg−1 ; frequency = 60–70 min−1 ) and were subjected to neuromuscular blockade with pancuronium bromide (2.5 mg kg−1 , I.V., Fresenius Kabi, Runcorn, UK). Partial pressure of end-tidal CO2 (P ETCO2 ) was monitored by a Capnogard CO2 sensor (Novametrix Medical Systems) placed on the expiratory line of the ventilator circuit. P ETCO2 was maintained at 50 mmHg  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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by adjusting the ventilator rate and/or inspired CO2 . A 2 cmH2 O positive end-expired pressure was applied by inserting the outlet tube of the ventilator 2 cm under the surface of the water. The bilateral phrenic nerves were isolated and sectioned distally in the cervical region by a ventral approach in all animals. In the second protocol (see below), the left phrenic nerve was subsequently de-sheathed and separated into small neurofilaments after confirmation of single fibre recording as previously described (Lee et al. 2009, 2013, 2015). The whole phrenic nerve and single phrenic fibre were placed on monopolar silver electrodes (#782500, A-M Systems, Carlsborg, WA, USA) connected to a differential A/C amplifier (amplification: 1000×; band-pass filter: 0.3–10 kHz, Model 1700, A-M Systems). All neural and blood pressure signals were digitized using a CED Power 1401 and stored in a computer with Spike 2 software. Experimental protocols

Two experimental protocols were conducted in the present study. In the first, bilateral phrenic nerves were recorded in seven uninjured and seven C2Hx rats. After stable recording of phrenic activity, two doses of capsaicin (1.0 and 1.5 µg kg−1 ) were delivered at approximately 20 min intervals through the right jugular vein catheter to activate bronchopulmonary C-fibres. To confirm that the capsaicin-induced reflexes mainly resulted from the activation of vagal bronchopulmonary C-fibre afferents, the cardiorespiratory response was evaluated with the same doses of capsaicin following a bilateral cervical vagotomy. In the second protocol, the right (i.e. uninjured side) whole phrenic nerve and left (i.e. injured side) single phrenic fibre were recorded in ten uninjured and 11 C2Hx rats. Once a stable recording of the whole phrenic nerve and phrenic fibre was established, a single dose of capsaicin (1.5 µg kg−1 ) was injected via the right jugular vein catheter to examine the response of individual phrenic motoneuron activity under the vagal-intact condition. At least a 20 min interval was allowed prior to examining the capsaicin-induced response in another single phrenic fibre. No more than two capsaicin injections were performed in a single rat during protocol 2. Spinal cord histology

After termination of the neurophysiological experiments, the C2Hx animals were euthanized via systemic perfusion with heparin-saline followed by 4% paraformaldehyde (Alfa Aesar, Ward Hill, MA, USA) and then 10% sucrose in 4% paraformaldehyde. The cervical spinal cord tissue was dissected and placed in 30% sucrose in PBS. After the tissue sank, the spinal cord was sectioned into 40 µm sections using a Cryostat (CM 1850, Leica, Wetzlar, Germany)

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and serially mounted on glass slides. The spinal cord sections were stained with cresyl violet (Acros Organics, Waltham, MA, USA), cover-slipped and evaluated with an upright microscope (DM750, Leica) connected to a digital camera (EOS 600D, Canon). The extent of ipsilateral spared white matter at the lesion epicentre was calculated by using ImageJ software, and the data are expressed as a percentage of the lateral and ventral white matter area of the contralateral side (Lee & Chang, 2014). A representative example of cervical spinal cord hemisection is presented in Fig. 1. The preserved spinal white matter in C2Hx animals was 9.4 ± 2.2% of the contralateral white matter. Data analysis and statistics Protocol 1. The respiratory cycle duration was calculated

by the rectified and smoothed (time constant: 25 ms) phrenic neurogram contralateral to the hemilesion. The inspiratory duration (TI ) was defined as the period between discharge onset of inspiratory phrenic bursting and the time point when the phrenic burst amplitude decreased by 50% of the maximum value (Lee et al. 2009). The expiratory duration (TE ) is calculated as the interval between the end of inspiration and the onset of subsequent inspiratory phrenic bursting. The respiratory frequency is calculated as 60 (TI + TE )−1 . The phrenic burst amplitude is defined as the difference between the maximum and minimum value of the processed phrenic neurogram within a single neural breath. These data are expressed as arbitrary units (a.u.) or percentages of baseline values (% BL) prior to capsaicin administration. The differences in inspiratory bursting onset between the contralateral and ipsilateral phrenic nerve were also calculated as previously described (Lee et al. 2013). The cardiorespiratory parameters were averaged over 10 s (baseline value) before capsaicin injection. Phrenic burst frequency and amplitude during the baseline

0.5 mm Figure 1. Representative example of a C2 spinal hemisection

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was analysed by a t test (uninjured vs. C2Hx animals) and two-way repeated measures analysis of variance (RM ANOVA) [factor one: group (uninjured vs. C2Hx animals); factor two: nerve (contralateral vs. ipsilateral)] followed by the Student–Newman–Keuls post hoc test, respectively. Bronchopulmonary C-fibre activation usually induces a complete cessation of phrenic bursting (i.e. apnoea) or a low amplitude of phrenic burst. We quantified the immediately phrenic response as ‘the period of robust phrenic inhibition’, which was defined as the period when the burst amplitude of the uninjured side phrenic nerve was < 20 % of baseline (Wilson & Bonham, 1997). The immediate cardiorespiratory responses (e.g. period of robust phrenic inhibition, bradycardia and hypotension) were analysed by two-way RM ANOVA [factor one: group (uninjured vs. C2Hx); factor two: condition (baseline vs. capsaicin)] followed by the Student–Newman–Keuls post hoc test. The amplitude of phrenic bursts immediately following a capsaicin-induced apnoea or robust inhibition was normalized to a percentage of the baseline values (% BL) and was compared by two-way RM ANOVA [factor one: group (uninjured vs. C2Hx animals); factor two: time point (baseline vs. breaths after capsaicin)]. In addition, linear regression analyses were used to analyse the relationship between bilateral phrenic burst amplitude after capsaicin administration. Discharge onset differences between the bilateral phrenic nerves after capsaicin treatment were averaged over ten breaths after capsaicin treatment and were analysed by two-way RM ANOVA [factor one: group (uninjured vs. C2Hx); factor two: condition (baseline vs. capsaicin)]. Protocol 2. The phrenic motoneuron type was classified based on the discharge onset time relative to the contralateral phrenic neurogram (Lee et al. 2009, 2013, 2015). The phrenic motoneuron that initiated bursting within the initial 20% of TI and > 20% of TI was classified as Early-I and Late-I, respectively. The firing behaviour of the phrenic motoneurons (e.g. discharge frequency, spike number per breath and discharge duration) was averaged over 10 s (baseline value) before capsaicin treatment. After capsaicin administration, the firing behaviours are expressed in individual breaths. The time course of capsaicin-induced phrenic motoneuron response was analysed by two-way RM ANOVA (factor one: phrenic motoneuron type; factor two: time points) followed by a Student–Newman–Keuls post hoc test. To further compare whether capsaicin induced differential inhibition on distinct phrenic motoneuron types, capsaicin-induced alterations of firing behaviours are expressed as percentages of baseline values and were averaged over 10 breaths after capsaicin injection. These data were also analysed by two-way RM ANOVA (factor one: phrenic motoneuron type; factor two: baseline vs. capsaicin  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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treatment) followed by a Student–Newman–Keuls post hoc test. All data are expressed as means ± standard errors. A P-value of less than 0.05 was considered significant.

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(P < 0.01). In addition, the onset of each burst on the ipsilateral phrenic nerve was always delayed relative to the onset on the contralateral phrenic nerve (Fig. 3). Bilateral phrenic nerve activity following intra-jugular capsaicin injection

Results Phrenic burst pattern following chronic cervical spinal cord injury

Representative examples of phrenic burst patterns are presented in Figs 2 and 3. Respiratory frequency was similar between uninjured (65 ± 1 min−1 ) and C2Hx (64 ± 1 min−1 ) animals during the baseline due to the entrainment effect of the ventilator. The phrenic burst pattern in uninjured animals was indistinguishable between the right (0.14 ± 0.01 a.u.) and left (0.17 ± 0.03 a.u.) phrenic nerve; however, C2Hx animals exhibited a significantly weaker burst amplitude in the ipsilateral (i.e. left, 0.03 ± 0.00 a.u.) compared to the contralateral (i.e. right, 0.28 ± 0.04 a.u.) phrenic nerve

Intra-jugular capsaicin administration caused pulmonary chemoreflexes characterized by a period of robust phrenic inhibition, hypotension and bradycardia in uninjured animals (Figs 2 and 4). Specifically, the period of robust inhibition of phrenic bursting was 2.2 ± 0.5 and 4.2 ± 0.8 s in response to 1.0 and 1.5 µg kg−1 capsaicin administration, respectively. This inhibitory period was significantly longer than the baseline expiratory duration (P < 0.01, Fig. 4A). However, the period of capsaicin-induced robust phrenic inhibition in C2Hx animals was significantly shorter than uninjured animals (P < 0.01, Fig. 4A). Both moderate (1.0 µg kg−1 ) and high (1.5 µg kg−1 ) doses of capsaicin induced significant hypotension and bradycardia in uninjured

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Figure 2. Representative examples of bilateral phrenic neurograms in response to intra-jugular capsaicin administration Intra-jugular capsaicin administration evoked a prolonged expiratory duration (i.e. apnoea) and mildly reduced the phrenic burst amplitude in uninjured animals; however, the same doses of capsaicin severely inhibited phrenic activity ipsilateral to the lesion. , rectified and smoothed signals. CL Phr and IL Phr represent the raw neural signals recorded from the contralateral and ipsilateral phrenic nerve, respectively. BP, blood pressure. The upward arrow represents the time point of capsaicin injection.  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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animals (P < 0.01, Fig. 4B, C), but C2Hx animals only demonstrated reduced heart rate in response to intra-jugular capsaicin administration (P < 0.01, Fig. 4C). The bilateral phrenic burst amplitude reached approximately 80% BL at the first breath after resuming from robust phrenic inhibition in uninjured animals (Figs 2 and 5). The phrenic burst amplitude of the contralateral phrenic nerve in C2Hx animals transiently decreased for three breaths after capsaicin (P < 0.05, Fig. 5B). However, intra-jugular capsaicin injection caused a preferential inhibition on the ipsilateral phrenic burst amplitude in C2Hx animals (Fig. 2). The ipsilateral phrenic burst amplitude was reduced significantly to 52 ± 9% BL and 40 ± 10% BL at first breath after 1.0 and 1.5 µg kg−1 capsaicin administration, respectively (P < 0.05, Fig. 5). This inhibition of phrenic activity was sustained for at least 10 breaths after apnoea. In uninjured animals, the response of bilateral phrenic burst amplitude was similar between the phrenic nerves. However, capsaicin injection caused a profound imbalance between the contralateral (i.e. uninjured) and ipsilateral (i.e. injured) phrenic activity in C2Hx animals (Figs 2 and 3). Based on linear regression analyses (Fig. 6), the ipsilateral phrenic burst amplitude remained attenuated even though the contralateral phrenic activity resumed to the baseline level. Additionally, capsaicin treatment also

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influenced the commencement of phrenic bursting. The onset of bursts occurred almost simultaneously on the left and right phrenic nerves in uninjured animals regardless of capsaicin administration (Figs 3 and 7). In C2Hx animals, the discharge onset of the ipsilateral phrenic nerve was later than that of the contralateral phrenic nerve, approximately 70 ms during baseline. This delay was exaggerated to 118 ± 21 and 149 ± 35 ms in response to moderate and high doses of capsaicin, respectively (P < 0.01, Fig. 7).

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IL Phr 0.5 s Figure 3. Representative examples demonstrating discharge onset of bilateral phrenic neurograms before and after capsaicin treatment The discharge onset of bilateral phrenic bursting is identical in uninjured animals before and after capsaicin injection. The ipsilateral phrenic nerve exhibited a later bursting onset compared to the contralateral phrenic nerve during the baseline. This onset difference was enhanced after capsaicin administration. , rectified and smoothed signals. CL Phr and IL Phr represent the raw neural signals recorded from the contralateral and ipsilateral phrenic nerve, respectively. The vertical line represents the onset of contralateral phrenic bursting. The vertical dotted line represents the discharge onset of ipsilateral phrenic nerve.

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Figure 4. Capsaicin-induced immediate cardiorespiratory responses Expiratory duration (A), mean arterial blood pressure (MAP, B) and heart rate (HR, C) were quantified during baseline and after two doses of capsaicin treatment. BL, baseline. ∗ P < 0.05; ∗∗ P < 0.01 compared with BL. ## P < 0.01 compared with uninjured animals.

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Phrenic motoneuron discharge pattern following chronic cervical spinal cord injury

Seventeen and 15 phrenic motoneurons were recorded during the baseline condition in uninjured and C2Hx animals, respectively. Representative examples of the discharge patterns of phrenic motoneurons in uninjured and C2Hx animals are presented in Figs 8–11. Eight Early-I and nine Late-I phrenic motoneurons were recorded in uninjured animals. The majority of phrenic motoneurons were Late-I type (n = 13) in C2Hx animals, and only one Early-I phrenic motoneuron was recorded. The discharge onset of Early-I phrenic motoneurons (49.3 ± 6.1 ms; 13.5 ± 1.7% TI ) in uninjured animals was expected to be earlier than the Late-I phrenic motoneurons in uninjured and C2Hx animals (P < 0.01). However, Late-I phrenic motoneurons had similar discharge onsets between the two groups [uninjured, 158 ± 13.6 ms (45.5 ± 3.7% TI ); C2Hx, 194 ± 17.0 ms (53.8 ± 4.5% TI )]. The discharge frequency was similar between Early-I (40.3 ± 1.8 Hz) and Late-I (37.0 ± 2.6 Hz) phrenic motoneurons in

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uninjured animals during baseline but the spike numbers per breath and discharge duration were significantly lower in Late-I than in Early-I phrenic motoneurons (P < 0.05, Fig. 12). In C2Hx animals, both discharge frequency (22.0 ± 2.5 Hz) and spike numbers (3 ± 0.5 per breath) of Late-I phrenic motoneurons were significantly lower than those in uninjured animals (P < 0.05, Fig. 12). Firing behaviour of phrenic motoneurons following intra-jugular capsaicin injection

Representative examples of phrenic motoneuron discharge patterns in response to intra-jugular capsaicin administration are presented in Figs 8–11. Intra-jugular capsaicin administration induced a robust inhibition of phrenic bursting, bradycardia and hypotension as expected. After the phrenic nerve recovered from capsaicin-induced robust inhibition, the firing behaviour of the phrenic motoneuron was differentially expressed across different phenotypes. For example, the discharge

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Figure 5. Time course of phrenic burst amplitude following capsaicin administration The contralateral (A, B) and ipsilateral (C, D) phrenic burst amplitudes were quantified during baseline and after capsaicin administration. Capsaicin injection induced a mild decline in contralateral phrenic activity but evoked a robust inhibition on ipsilateral phrenic bursting in C2Hx animals. The y-axis represents the rectified and smoothed phrenic burst amplitude. The data are expressed as a percentage of the baseline value (% BL). ∗ P < 0.05 compared with BL. # P < 0.05 significant difference between uninjured vs. C2Hx animals.  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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frequency of Early-I phrenic motoneurons in uninjured animals was not significantly influenced by capsaicin treatment; however, their spikes and discharge duration were attenuated for several breaths after capsaicin treatment (P < 0.05, Fig. 12). The discharge frequency of Late-I phrenic motoneurons in uninjured animals was significantly decreased from the first (10.8 ± 8.2 Hz) to fifth (15.2 ± 6.4 Hz) breaths following capsaicin treatment (P < 0.05, Fig. 12). Both spikes and discharge durations were decreased for approximately 10 breaths following capsaicin administration. The firing behaviours of Late-I phrenic motoneurons in C2Hx animals were

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severely inhibited by capsaicin treatment (Figs 10 and 11). A portion of Late-I phrenic motoneurons (5/13) in C2Hx rats even ceased firing for at least 10 breaths following capsaicin injection (Fig. 11). The mean data demonstrated that the discharge frequency decreased from 22.0 ± 2.5 to 6.1 ± 3.6 Hz at the first breath and to 11.7 ± 3.7 Hz at the tenth breath after capsaicin treatment (P < 0.05, Fig. 12). The spike numbers of Late-I phrenic motoneurons (3.0 ± 0.5) in C2Hx animals were low during baseline; therefore, these motoneurons only exhibited 1–2 spikes following capsaicin treatment. Moreover, the discharge duration was profoundly reduced in response to capsaicin administration (P < 0.05, Fig. 12). To further investigate whether distinct phrenic motoneuron types respond differentially to intra-jugular capsaicin administration, firing behaviours (discharge frequency, spike numbers per breath, discharge duration) were averaged over 10 breaths after capsaicin treatment and were expressed as a percentage of the baseline value. The data analyses demonstrated that Early-I

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Figure 7. The mean difference in discharge onset between contralateral and ipsilateral inspiratory phrenic bursting The relative onset difference between contralateral and ipsilateral phrenic bursting was quantified during baseline and after 1.0 µg kg−1 (A) and 1.5 µg kg−1 (B) capsaicin injection. ∗∗ P < 0.01 compared with baseline. ## P < 0.01 significant difference between uninjured and C2Hx animals.  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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phrenic motoneurons of uninjured animals were relatively resistant to the inhibitory effects of capsaicin. Thus, capsaicin administration caused a greater inhibition on Late-I compared to Early-I phrenic motoneurons in uninjured animals (P < 0.01, Fig. 13). Although the discharge frequency and spikes of Late-I phrenic motoneurons were lower in C2Hx animals during the baseline, the percentage reduction in these firing parameters after capsaicin treatment was similar between uninjured and C2Hx animals. Discussion The present study demonstrated that the inhibitory effect of capsaicin on ipsilateral phrenic burst amplitude was exaggerated after unilateral cervical spinal cord injury. In

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addition, intra-jugular capsaicin administration caused an imbalance in burst amplitude and discharge onset between contralateral vs. ipsilateral phrenic activity in chronic C2Hx animals. The altered phrenic response to capsaicin treatment during the chronic injury phase was due to changes in phrenic motoneuron phenotype and discharge pattern. Specifically, the delayed discharge onset of ipsilateral phrenic bursting in C2Hx animals resulted from a lower proportion of Early-I phrenic motoneurons. Moreover, the severe reduction in ipsilateral phrenic burst amplitude following capsaicin injection was partially caused by a higher proportion of Late-I phrenic motoneurons, which were relatively sensitive to capsaicin treatment compared to Early-I phrenic motoneurons. Lastly, the Late-I phrenic motoneurons in C2Hx animals had a slower recovery time course in response to capsaicin

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PhrMN 0.2 s Figure 8. A representative example of Early-I phrenic motoneuron bursting in an uninjured animal A, Early-I phrenic motoneuron bursting before and after intra-jugular capsaicin administration. B, the expanded time scale data trace from a single breath labelled by a and b in A. BP, blood pressure; , rectified and smoothed signals. CL Phr represents the raw neural signals recorded from the contralateral phrenic nerve. Mean f, mean discharge frequency of phrenic motoneurons (bin: 100 ms); PhrMN, raw signals of phrenic motoneuron activity.  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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administration. These results suggest that the phrenic motoneuron phenotype and discharge following C2Hx leads to alterations of whole bursting in response to bronchopulmonary activation.

shift in pattern phrenic C-fibre

Response of phrenic motoneuron firing following bronchopulmonary C-fibre activation

Although bronchopulmonary C-fibre-evoked pulmonary chemoreflexes have been extensively reported in many previous studies (Lin & Lee, 2002; Lee et al. 2003; Lee & Chang, 2014; Tsai & Lee, 2014; Lin et al. 2015), the influence of capsaicin-induced bronchopulmonary C-fibre activation on phrenic motoneuron bursting is poorly understood. The current experiment demonstrated that intra-jugular capsaicin administration differentially

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modulated the firing behaviours of Late-I vs. Early-I phrenic motoneurons in uninjured animals. In particular, the discharge frequency of Late-I but not Early-I motoneurons was inhibited following capsaicin treatment. The recovery time course of spikes and discharge duration are slower in Late-I vs. Early-I phrenic motoneurons. The factors contributing to these differential responses of phrenic motoneurons could occur at both the supraspinal and the spinal levels. Monteau et al. (1985) indicated that inspiratory bursting of Early-I and Late-I phrenic motoneurons was triggered by different premotor neurons in the brainstem in the cat. Intracellular recording experiments demonstrated that depolarizing shifts of the membrane potential during inspiration in early-onset phrenic motoneurons is higher than for other motoneuron types, suggesting that the central inspiratory drive is different between motoneuron types (Hayashi & Fukuda,

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PhrMN 0.2 s Figure 9. A representative example of Late-I phrenic motoneuron bursting in an uninjured animal A, a Late-I phrenic motoneuron bursting before and after intra-jugular capsaicin administration. B, the expanded time scale data trace from a single breath labelled by a and b in A. Abbreviations are as defined in Fig. 8.  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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1995). Wilson & Bonham (1997) further demonstrated that these inspiratory bulbospinal neurons could be inhibited by the activation of bronchopulmonary C-fibres. These results lead us to suspect that distinct responses of Early-I vs. Late-I neurons are partially due to differential inhibition of bronchopulmonary C-fibre activation on their premotor neurons. In addition, Hayashi & Fukuda (1995) demonstrated that late-onset phrenic motoneurons have a relative lower input resistance and higher rheobase. These intrinsic membrane properties may cause late-onset phrenic motoneurons to have higher susceptibilities to reduce discharge frequency when inspiratory drives are decreased by bronchopulmonary C-fibre activation. Lastly, there is a small population of pre-phrenic interneurons located at the cervical spinal cord (Lane et al. 2008; Buttry & Goshgarian, 2014). These interneurons can respond to respiratory challenges and send projections to phrenic motoneurons (Lane et al. 2009; Sandhu

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et al. 2015). Hence, these pre-phrenic interneurons may also be involved in the regulation of divergent phrenic motoneuron responses. Alteration of the pulmonary chemoreflex following chronic cervical spinal cord injury

The present study has demonstrated that intra-jugular capsaicin administration caused a complete cessation of phrenic bursting (Fig. 9) or resulted in a period of extremely low phrenic burst amplitude (Figs 2, 8 and 10). These phrenic responsive patterns have also been observed in other studies using anaesthetized and ventilated rats (Wilson & Bonham, 1997; Moreira et al. 2007). Wilson & Bonham (1997) recorded central respiratory neurons within the brainstem and found that bronchopulmonary C-fibre activation can specifically excite decrementing expiratory neurons and inhibit inspiratory

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PhrMN 0.2 s Figure 10. A representative example of Late-I phrenic motoneuron bursting in a C2Hx animal A, Late-I phrenic motoneuron bursting before and after intra-jugular capsaicin administration. B, the expanded time scale data trace from a single breath labelled by a and b in A. Abbreviations are as defined in Fig. 8.  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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neurons. Similarly, Paton (1997) demonstrated that some post-inspiratory neurons of the ventral respiratory group were depolarized during bronchopulmonary C-fibre activation. Both studies indicated that alteration of central respiratory neuronal activity was coincident with bronchopulmonary C-fibre activation-induced changes of respiratory cycle (Paton, 1997; Wilson & Bonham, 1997). In addition, activation of bronchopulmonary C-fibres can suppress the neuronal activity of central chemoreceptors (e.g. retrotrapezoid nucleus), which usually provides an excitatory drive to the respiratory networks (Moreira et al. 2007). Moreover, our previous reports demonstrated that inspiratory hypoglossal activity was eliminated during the period of capsaicin-induced robust

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phrenic inhibition (Lee et al. 2007). Taken together, these data lead us to speculate that the period of capsaicin-induced cessation or inhibition of phrenic bursting was mainly contributed by alteration of supraspinal respiratory activity. Accordingly, attenuation of capsaicin-induced apnoea or robust phrenic inhibition in C2Hx animals may partially reflect alteration of supraspinal respiratory sensitivity to bronchopulmonary C-fibre activation following spinal cord injury. In addition, cervical spinal cord injury usually alters the breathing pattern, which may influence the respiratory mechanics and/or ventilation–perfusion relationship. We cannot exclude that the respiratory drives may be different in C2Hx versus uninjured animals even controlling for

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PhrMN 0.2 s Figure 11. A representative example illustrating capsaicin-induced cessation of Late-I phrenic motoneuron bursting in a C2Hx animal A, bursting of Late-I phrenic motoneurons was completely abolished by capsaicin administration for several breaths following apnoea. B, the expanded time scale data trace from a single breath labelled by a and b in A. Abbreviations are as defined in Fig. 8.  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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P ETCO2 . Therefore, the alteration of pulmonary chemoreflex in C2Hx animals may be also contributed by changes of chemoreceptor drives. The current study observed that not all responses induced by intra-jugular capsaicin administration were attenuated. For example, capsaicin produced a greater and longer inhibition of ipsilateral phrenic activity in C2Hx compared to uninjured animals. Moreover,

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the discharge onset of ipsilateral phrenic bursting was delayed after capsaicin treatment in C2Hx animals. The distinct response of respiratory activity after cervical spinal cord injury was also observed following intermittent hypoxic treatment. Doperalski & Fuller (2006) demonstrated that intermittent hypoxia-induced long-term facilitation in ipsilateral phrenic amplitude was augmented but long-term facilitation in respiratory frequency was instead blunted in C2Hx rats compared to uninjured controls. Furthermore, blockade of bulbospinal respiratory pathways to phrenic motoneurons only

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BL 1 2 3 4 5 6 7 8 9 10 Breaths post-capsaicin Figure 12. Time course of changes in firing behaviours of phrenic motoneurons following capsaicin administration Discharge frequency (A), spike number per breath (B) and discharge duration (C) were quantified during baseline and after capsaicin administration. ∗ P < 0.05; ∗∗ P < 0.01 compared with BL. a, P < 0.05 vs. Early-I of uninjured animals; b, vs. Late-I of uninjured animals.  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

C2Hx_Late-I Figure 13. Differential inhibition of intra-jugular capsaicin injection on firing behaviours of distinct phrenic motoneuron types Response of discharge frequency (A), spike number per breath (B) and discharge duration (C) following intra-jugular capsaicin injection are presented as a percentage of baseline (% BL) ∗∗ P < 0.01 compared with BL (i.e. 100% BL). ## P < 0.01 vs. Early-I of uninjured animals.

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evoked a facilitation of phrenic burst amplitude but not respiratory frequency (Streeter & Baker-Herman, 2014). Inactivity-induced facilitation in phrenic burst frequency and amplitude was also differentially expressed (Baertsch & Baker-Herman, 2013). These results suggest that supraspinal (e.g. respiratory frequency) and spinal respiratory (e.g. phrenic burst amplitude) activity was differentially influenced by the experimental inactivation of the respiratory pathways. Further studies are necessary to investigate the mechanism underlying supraspinal and spinal respiratory neuroplasticity following spinal cord injury. Motoneuron mechanisms underlying altered phrenic responses to bronchopulmonary C-fibre activation

Because the capsaicin-induced apnoeic response was attenuated in C2Hx animals, the robust inhibition of the ipsilateral phrenic activity following capsaicin administration should not be due to overexcitation of bronchopulmonary C-fibre activation. Our current data demonstrated that the phenotype population and discharge pattern of the phrenic motoneurons were changed following chronic C2Hx. The phrenic motoneuron discharge pattern is controlled by extrinsic synaptic inputs and intrinsic motoneuron properties (Monteau et al. 1985; Lee & Fuller, 2011; Seven et al. 2014). The inspiratory bursting of phrenic motoneurons is triggered by premotor neurons in the rostral ventral respiratory group, and some premotor neurons send projections across the spinal midline. These neurons then innervate the contralateral phrenic motoneurons (Ellenberger & Feldman, 1988) and contribute to recovery of phrenic bursting after C2Hx through activation of crossed spinal pathways (Goshgarian et al. 1991). Accordingly, C2Hx may alter the activity in these bulbospinal premotor neurons and thereby cause a change in the phrenic nerve response to bronchopulmonary C-fibre activation. Moreover, our lab and others have demonstrated that the distribution of phrenic motoneuron type was dominated by Late-I phrenic motoneurons after lateral cervical spinal injury (El-Bohy & Goshgarian, 1999; Lee et al. 2013, 2015), suggesting that the crossed phrenic pathway may primarily activate Late-I phrenic motoneurons. Because intra-jugular capsaicin preferentially inhibits Late-I phrenic motoneurons in uninjured rats, the higher proportion of Late-I phrenic motoneurons in C2Hx animals may lead to a greater inhibition on ipsilateral phrenic burst amplitude following chronic C2Hx. In addition, the activity of Late-I phrenic motoneurons was weaker in C2Hx than in uninjured animals. We suspect that the lower baseline activity may increase the susceptibility of motoneurons to inhibitory inputs. Collectively, influences on the amplitude of phrenic

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nerve bursts could occur via actions at either the spinal or the supraspinal level. Physiological significance

The pulmonary chemoreflex is a critical defence reflex to modulate the cardiorespiratory pattern in response to the inhalation of irritants (Lee & Pisarri, 2001). Although the attenuation of the respiratory component of the pulmonary chemoreflex (i.e. robust inhibition of phrenic bursting) may prevent further reductions of breathing capacity, it can also increase the susceptibility to inhaled irritants. Clinical reports have demonstrated that inspiratory loading-induced inhibition of inspiratory muscle activity was only observed in a few patients (2/14, 14%) with cervical spinal injury (McBain et al. 2015). Other defensive respiratory behaviours (e.g. augmented breath and cough) are also impaired after spinal cord injury (Bolser et al. 2009). These findings suggest that the higher risk of aspiration in subjects with cervical spinal cord injury may be due to the attenuation of multiple protective reflexes. Bronchopulmonary C-fibres are the primary chemosensitive afferents in the lung and can be activated by several inflammatory mediators (such as tumour necrosis factor-alpha and interleukin-1 beta) (Yu et al. 2007; Lin et al. 2013). Clinically, pulmonary complications are always associated with cervical spinal cord injury and are risk factors for long-term mechanical ventilation (Winslow & Rozovsky, 2003; Roquilly et al. 2014). These pulmonary pathological conditions may influence bronchopulmonary C-fibre activity and, in turn, impact respiratory motor outputs. In addition, Huxtable et al. (2013) indicated that systemic inflammation can attenuate the expression of respiratory neuroplasticity in the rat. Our current data demonstrated that phrenic bursting ipsilateral to the lesion is substantially inhibited during capsaicin-induced bronchopulmonary C-fibre activation. Therefore, we suspect that the recovery of phrenic motor outputs may be retarded during pulmonary complications following cervical spinal cord injury. Accordingly, the prevention and alleviation of lung inflammation in subjects with spinal cord injury may improve respiratory motor and lung functions. References Baertsch NA & Baker-Herman TL (2013). Inactivity-induced phrenic and hypoglossal motor facilitation are differentially expressed following intermittent vs. sustained neural apnea. J Appl Physiol (1985) 114, 1388–1395. Bolser DC, Jefferson SC, Rose MJ, Tester NJ, Reier PJ, Fuller DD, Davenport PW & Howland DR (2009). Recovery of airway protective behaviours after spinal cord injury. Respir Physiol Neurobiol 169, 150–156.

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Butler JE, Hudson AL & Gandevia SC (2014). The neural control of human inspiratory muscles. Prog Brain Res 209, 295–308. Buttry JL & Goshgarian HG (2014). Injection of WGA-Alexa 488 into the ipsilateral hemidiaphragm of acutely and chronically C2 hemisected rats reveals activity-dependent synaptic plasticity in the respiratory motor pathways. Exp Neurol 261, 440–450. Doperalski NJ & Fuller DD (2006). Long-term facilitation of ipsilateral but not contralateral phrenic output after cervical spinal cord hemisection. Exp Neurol 200, 74–81. El-Bohy AA & Goshgarian HG (1999). The use of single phrenic axon recordings to assess diaphragm recovery after cervical spinal cord injury. Exp Neurol 156, 172–179. Ellenberger HH & Feldman JL (1988). Monosynaptic transmission of respiratory drive to phrenic motoneurons from brainstem bulbospinal neurons in rats. J Comp Neurol 269, 47–57. Fuller DD, Doperalski NJ, Dougherty BJ, Sandhu MS, Bolser DC & Reier PJ (2008). Modest spontaneous recovery of ventilation following chronic high cervical hemisection in rats. Exp Neurol 211, 97–106. Fuller DD, Golder FJ, Olson EB Jr & Mitchell GS (2006). Recovery of phrenic activity and ventilation after cervical spinal hemisection in rats. J Appl Physiol (1985) 100, 800–806. Goshgarian HG (2009). The crossed phrenic phenomenon and recovery of function following spinal cord injury. Respir Physiol Neurobiol 169, 85–93. Goshgarian HG, Ellenberger HH & Feldman JL (1991). Decussation of bulbospinal respiratory axons at the level of the phrenic nuclei in adult rats: a possible substrate for the crossed phrenic phenomenon. Exp Neurol 111, 135–139. Hayashi F & Fukuda Y (1995). Electrophysiological properties of phrenic motoneurons in adult rats. Jpn J Physiol 45, 69–83. Hsu SH & Lee KZ (2015). Effects of serotonergic agents on respiratory recovery after cervical spinal injury. J Appl Physiol (1985) 119, 1075–1087. Huxtable AG, Smith SM, Vinit S, Watters JJ & Mitchell GS (2013). Systemic LPS induces spinal inflammatory gene expression and impairs phrenic long-term facilitation following acute intermittent hypoxia. J Appl Physiol (1985) 114, 879–887. Kong FJ & Berger AJ (1986). Firing properties and hypercapnic responses of single phrenic motor axons in the rat. J Appl Physiol (1985) 61, 1999–2004. Lane MA (2011). Spinal respiratory motoneurons and interneurons. Respir Physiol Neurobiol 179, 3–13. Lane MA, Lee KZ, Fuller DD & Reier PJ (2009). Spinal circuitry and respiratory recovery following spinal cord injury. Respir Physiol Neurobiol 169, 123–132. Lane MA, White TE, Coutts MA, Jones AL, Sandhu MS, Bloom DC, Bolser DC, Yates BJ, Fuller DD & Reier PJ (2008). Cervical prephrenic interneurons in the normal and lesioned spinal cord of the adult rat. J Comp Neurol 511, 692–709. Lee KZ & Chang YS (2014). Recovery of the pulmonary chemoreflex and functional role of bronchopulmonary C-fibres following chronic cervical spinal cord injury. J Appl Physiol (1985) 117, 1188–1198.  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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Lee KZ, Dougherty BJ, Sandhu MS, Lane MA, Reier PJ & Fuller DD (2013). Phrenic motoneuron discharge patterns following chronic cervical spinal cord injury. Exp Neurol 249, 20–32. Lee KZ & Fuller DD (2011). Neural control of phrenic motoneuron discharge. Respir Physiol Neurobiol 179, 71–79. Lee KZ, Fuller DD, Lu IJ, Lin JT & Hwang JC (2007). Neural drive to tongue protrudor and retractor muscles following pulmonary C-fibre activation. J Appl Physiol 102, 434–444. Lee KZ, Huang YJ & Tsai IL (2014). Respiratory motor outputs following unilateral midcervical spinal cord injury in the adult rat. J Appl Physiol (1985) 116, 395–405. Lee KZ, Reier PJ & Fuller DD (2009). Phrenic motoneuron discharge patterns during hypoxia-induced short-term potentiation in rats. J Neurophysiol 102, 2184–2193. Lee KZ, Sandhu MS, Dougherty BJ, Reier PJ & Fuller DD (2010). Influence of vagal afferents on supraspinal and spinal respiratory activity following cervical spinal cord injury in rats. J Appl Physiol (1985) 109, 377–387. Lee KZ, Sandhu MS, Dougherty BJ, Reier PJ & Fuller DD (2015). Hypoxia triggers short term potentiation of phrenic motoneuron discharge after chronic cervical spinal cord injury. Exp Neurol 263, 314–324. Lee LY & Pisarri TE (2001). Afferent properties and reflex functions of bronchopulmonary C-fibres. Respir Physiol 125, 47–65. Lee LY, Shuei Lin Y, Gu Q, Chung E & Ho CY (2003). Functional morphology and physiological properties of bronchopulmonary C-fibre afferents. Anat Rec A Discov Mol Cell Evol Biol 270, 17–24. Lin RL, Lin YJ, Geer MJ, Kryscio R & Lee LY (2013). Pulmonary chemoreflex responses are potentiated by tumor necrosis factor-alpha in mice. J Appl Physiol (1985) 114, 1536–1543. Lin YJ, Lin RL, Ruan T, Khosravi M & Lee LY (2015). A synergistic effect of simultaneous TRPA1 and TRPV1 activations on vagal pulmonary C-fibre afferents. J Appl Physiol (1985) 118, 273–281. Lin YS & Lee LY (2002). Stimulation of pulmonary vagal C-fibres by anandamide in anaesthetized rats: role of vanilloid type 1 receptors. J Physiol 539, 947–955. McBain RA, Hudson AL, Gandevia SC & Butler JE (2015). Short-latency inhibitory reflex responses to inspiratory loading of the scalene muscles are impaired in spinal cord injury. Exp Physiol 100, 216–225. Monteau R, Khatib M & Hilaire G (1985). Central determination of recruitment order: intracellular study of phrenic motoneurons. Neurosci Lett 56, 341–346. Moreira TS, Takakura AC, Colombari E & Guyenet PG (2007). Activation of 5-hydroxytryptamine type 3 receptor-expressing C-fibre vagal afferents inhibits retrotrapezoid nucleus chemoreceptors in rats. J Neurophysiol 98, 3627–3637. Navarrete-Opazo A, Vinit S, Dougherty BJ & Mitchell GS (2015). Daily acute intermittent hypoxia elicits functional recovery of diaphragm and inspiratory intercostal muscle activity after acute cervical spinal injury. Exp Neurol 266C, 1–10. Paton JF (1997). Rhythmic bursting of pre- and post-inspiratory neurones during central apnoea in mature mice. J Physiol 502, 623–639.

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Additional information Competing interests None declared. Author contributions K.-Z.L. contributed to all aspects of the present study. Funding Support for this work was provided by grants from the Ministry of Science and Technology (Most 102-2320-B-110-004-MY3), National Health Research Institutes (NHRI-EX105-10223NC) and NSYSU-KMU Joint Research Project (105-I004).

 C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society