Expiratory-modulated laryngeal motoneurons exhibit a hyperpolarization preceding depolarization during superior laryngeal nerve stimulation in the in vivo adult rat

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Expiratory-modulated laryngeal motoneurons exhibit a hyperpolarization preceding depolarization during superior laryngeal nerve stimulation in the in vivo adult rat Tara G. Bautista, Qi-Jian Sun, Paul M. Pilowsky⁎ Australian School of Advanced Medicine, L1 F10A, Macquarie University, NSW 2109, Australia

A R T I C LE I N FO

AB S T R A C T

Article history:

Swallowing requires the sequential activation of tongue, pharyngeal and esophageal

Accepted 15 January 2012

muscles to propel the food bolus towards the stomach. Aspiration during swallow is

Available online 24 January 2012

prevented by adduction of the vocal cords during the oropharyngeal phase. Expiratorymodulated laryngeal motoneurons (ELM) exhibit a burst of action potentials during swal-

Keywords:

lows elicited by electrical stimulation of the superior laryngeal nerve (SLN). Here we sought

Laryngeal motoneuron

to investigate changes in membrane potential in ELM during superior laryngeal nerve stim-

Superior laryngeal nerve stimulation

ulation in the anaesthetised, in vivo adult rat preparation. Intracellular recordings of ELM in

Swallow

the caudal nucleus ambiguus (identified by antidromic activation from the recurrent laryn-

Intracellular recording

geal nerve) demonstrated that ELM bursting activity following SLN stimulation is associated

Rat

with a depolarization that is preceded by a small hyperpolarization. During spontaneous

Hyperpolarization

ELM bursts, the preceding hyperpolarization separated the bursting activity from its usual post-inspiratory activity. These findings demonstrate that the in vivo adult rat preparation is suitable for the study of swallow-related activity in laryngeal motoneurons. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1.

Introduction

During swallow, inadvertent aspiration of food is prevented by apnea and the protective actions of the larynx. Within the larynx, contraction of laryngeal adductor muscles brings the vocal cords together to close the glottis (Doty and Bosma, 1956). In addition, the food bolus is diverted from the laryngeal vestibule and respiratory tract by elevation and tilting of the larynx in the anterior direction, as well as by lowering of the epiglottis (Logemann et al., 1992; Shaker et al., 1990). Motor deficits and an inability to temporally coordinate swallows with breathing are both associated with a higher risk of

aspiration in patients with neurodegenerative diseases, such as motoneuron disease, Steele–Richardson syndrome and Parkinson's disease, in whom aspiration pneumonia is a common cause of death (Gross et al., 2008). A recent publication reported that laryngeal dysfunction is a prominent feature in a transgenic mouse model for tauopathic neurodegenerative disease (Dutschmann et al., 2010). The sequential wave of muscular contraction in the upper airway (UA) and esophagus to simultaneously deliver the food bolus to the stomach and prevent aspiration is coordinated by inputs from the swallow central pattern generator (Jean, 2001; Miller, 1982). Studies in cat and sheep demonstrate

⁎ Corresponding author. Fax: + 61 2 9812 3600. E-mail address: [email protected] (P.M. Pilowsky). Abbreviations: EPSP, Excitatory post-synaptic potential; ELM, expiratory-modulated laryngeal motoneuron; IPSP, inhibitory postsynaptic potential; RLN, recurrent laryngeal nerve; SLN, superior laryngeal nerve; UA, upper airway 0006-8993/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2012.01.037

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Intracellular recordings were obtained from 7 ELM in 6 rats. Recorded neurons were typically found in the region ±0.5 mm rostro-caudal, and 1.8–2 mm lateral, to the calamus scriptorius, and 1.7–2.1 mm deep to the dorsal surface of the medulla. Neurons were identified as ELM if they exhibited: 1) antidromic excitation from the ipsilateral recurrent laryngeal nerve (RLN) and a positive collision test, and; 2) abrupt depolarization during the post-inspiratory phase and hyperpolarization during the inspiratory phase (Figs. 1A, 2A, 3). Antidromic excitation from the RLN was observed as an action potential occurring at a constant latency of 2.4 ± 0.8 ms that was abolished if it collided with a spontaneous action potential occurring within this amount of time prior to the stimulus (Fig. 1B). The average membrane potential of recorded neurons was −66.9 ± 12.2 mV with a maximum difference in membrane potential of 8.1 ± 3.1 mV between the post-inspiratory depolarization and inspiratory hyperpolarization.

frequencies of SLN stimulation (Figs. 1B, 2A). The average latency of orthodromic action potentials was 6.2 ± 0.7 ms when present. Orthodromic action potentials could be differentiated from antidromic action potentials as they were associated with a preceding EPSP, synaptic ‘jitter’ and an obvious afterhyperpolarization (Fig. 1B). High frequency (20 Hz) SLN stimulation resulted in a prolonged inhibition of inspiratory phrenic nerve discharge (apnea) concurrent with a prolonged depolarization and vigorous firing in ELM (Fig. 2B). The stimulus trains were started at random with the phrenic cycle and always terminated an inspiratory-related discharge prematurely if stimulation began mid-inspiration. The average membrane depolarization of ELM during the entire stimulation period was 3.4 ± 1.7 mV. The firing activity in ELM comprised of short high frequency bursts that were difficult to discriminate from the numerous orthodromic action potentials. Robust ELM firing and decreased phrenic nerve discharge frequency often continued beyond the stimulation period. (~10 s). Basal phrenic nerve discharge frequency and normal post-inspiratory activity in ELM was re-established within 10 s after the stimulus ended. In comparison, well-defined bursting activity in ELM was observed using lower frequencies of SLN stimulation (Figs. 1A, 2A). ELM bursting activity typically occurred along with a plateaulike depolarization and each action potential occurred following an EPSP (see also Fig. 3). At 5 and 10 Hz SLN stimulation, ELM bursts were often associated with ‘quantal misses’ in the subsequent inspiratory-related phrenic nerve discharge, as predicted by the (1:1) entrainment of phrenic nerve discharge with the rate of mechanical ventilation (Sun et al., 2011b). Occasionally, ELM bursts coincided with small amplitude phrenic nerve discharges (‘swallow breaths’). Swallow breaths were significantly reduced in amplitude, and often duration, compared to inspiratory-related phrenic nerve discharges occurring prior to the stimulus train. These swallow-related phrenic nerve discharges were distinct from prematurely terminated inspiratory-related phrenic discharges, which if present, occurred before ELM bursts. During lower frequencies of SLN stimulation, almost all ELM bursts were preceded by a small hyperpolarization of short duration (mean of all bursts: 6.1 ± 2.5 mV). The nadir of the preceding hyperpolarization consistently occurred ~0.03 s prior to the start of the ELM burst. However, the hyperpolarization was noticeably absent in a small number of bursts. In these instances, a stimulus was delivered at the same time during which the hyperpolarization was expected; however, neither the expected hyperpolarization nor the orthodromic action potential was observed. The depolarization during ELM bursts was typically followed by a slow decrease in membrane potential that was similar to the decline in membrane potential that ELM exhibit during late expiration. The characteristics of ELM bursts (mean depolarization, mean frequency and duration of bursts, magnitude of preceding hyperpolarization) were not statistically different during 5 and 10 Hz stimulation (Table 1).

2.1.

2.2.

that swallow is associated with both excitatory and inhibitory events within the cranial motoneurons, which supply UA and esophageal muscles (Tomomune and Takata, 1988; Zoungrana et al., 1997). Intracellular recordings of pharyngeal and esophageal motoneurons in the rostral nucleus ambiguus reveal excitatory post-synaptic potentials (EPSPs) and depolarization during swallow elicited by very brief high frequency stimulation of the superior laryngeal nerve (SLN) (Zoungrana et al., 1997). In addition, inhibitory post-synaptic potentials (IPSPs) and hyperpolarization are often observed preceding excitatory swallow-related activities in these cranial motoneurons. Motoneurons supplying laryngeal adductor muscles, which are normally active in the post-inspiratory/early expiratory period of the respiratory cycle, also show brief hyperpolarization prior to a burst of action potentials during swallow in cat (Gestreau et al., 2000; Numasawa et al., 2004; Shiba et al., 1999; Suzuki et al., 2010). The inhibitory events are thought to delay excitement of motoneurons to enable an orderly spatiotemporal contraction of muscles. Swallowrelated inhibition may also regulate motoneuronal excitability and contribute to depolarization by a post-inhibitory rebound mechanism. The objective of the present study was to investigate changes in membrane potential of expiratory-modulated laryngeal motoneurons (ELM) of the caudal nucleus ambiguus that supply the primary laryngeal adductor muscles during swallow elicited by SLN stimulation in the in vivo rat. We seek to determine if swallow-related activity in rat ELMs is similar to other species, given that there are subtle speciesrelated differences in swallow-related laryngeal adductor muscle activity (Doty and Bosma, 1956). Furthermore, it is important to investigate upper airway reflexes in rat because, unlike cat and other species, they do not exhibit complex oropharyngeal behaviours, such as cough and vomit.

2.

Results

ELM behaviour during SLN stimulation

Five of the 7 ELM exhibited orthodromic action potentials during SLN stimulation that were more common with higher

Spontaneous ELM bursts

Spontaneous ELM bursts following SLN stimulation were also observed and were similar in appearance to bursts occurring

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Fig. 1 – Responses to 5 Hz train stimulation of the superior laryngeal nerve (SLN). A) An intracellular recording from an expiratory-modulated laryngeal motoneuron (ELM) in relation to integrated phrenic nerve activity (PNA) during SLN stimulation (5 s; 1–1.5 × threshold voltage to produce apnea; 0.2 ms pulse duration). Two distinct ELM responses are observed: orthodromic action potentials following single stimuli (grey box) and high frequency bursts. ELM bursts were noticeably preceded by a small hyperpolarization (dots) and often followed by ‘misses’ in the next expected phrenic nerve discharge (arrowheads). B) In the left box, antidromic stimulation from the recurrent laryngeal nerve. A constant latency action potential (2.4 ms) following each stimulus is observed (left trace; 5 overlain traces aligned by stimulus artefact denoted by triangle). This was abolished if it collided with a spontaneous action potential occurring within 2.4 ms prior to the stimulus (arrowhead; right trace). In the right box, expanded trace of overlain orthodromic action potentials within the grey box in A. Note the presence of small excitatory post-synaptic potential (EPSP; open arrowhead) and synaptic 'jitter' associated with the orthodromic action potential onset compared with the absence of these features in the antidromic action potential.

during stimulus trains (Fig. 3). In such bursts, the preceding small hyperpolarization was more prominently observed (7.4 ± 1.7 mV; n = 9). Spontaneous ELM bursts occurred during early expiration and consistently associated with a ‘swallow-

breath’ (n= 9/9). In addition, the subsequent inspiratoryrelated phrenic nerve discharge was absent following spontaneous ELM bursts. Importantly, spontaneous ELM bursts and depolarization were distinctly separate from their normal

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post-inspiratory activity (Fig. 3). The post-inspiratory depolarization was always terminated prematurely by burst-related hyperpolarization (Fig. 3). The mean latency of spontaneous ELM

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bursts from the peak of the preceding inspiratory-related phrenic nerve discharge was 170.4± 85.6 ms (range: 43.4– 284.1 ms).

Fig. 2 – Responses to 10 and 20 Hz train stimulation of the SLN. A) Several ELM bursts occurred with higher frequency SLN stimulation. Most were associated with a preceding small hyperpolarization (dots) although the asterisk denotes an example without. A phrenic apnea develops as a result of multiple misses in phrenic nerve discharge (arrowheads). A spontaneous ELM burst accompanied by a small amplitude phrenic nerve discharge (‘swallow-breath’) following the train stimulation is seen on the far right. B) 20 Hz SLN stimulation elicits depolarization and strong firing activity in the recorded ELM that continues beyond the stimulus train.

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neurons were not classified as inspiratory-modulated laryngeal motoneurons as they did not exhibit antidromic excitation in response to RLN or SLN stimulation. Two of these neurons exhibited a ramp-like depolarization starting midexpiration and firing activity that reached peak levels coincident with the peak in inspiratory-related phrenic nerve discharge: all 3 neurons were then sharply hyperpolarized. One neuron exhibited an augmenting depolarization that closely mirrored the ramping activity in inspiratory-related phrenic nerve discharge (Fig. 4). Its membrane potential exhibited repolarization concomitant with the waning phrenic nerve discharge in the post-inspiratory phase. The nadir in membrane potential in all 3 inspiratory-modulated neurons occurred in late expiration. During low frequency SLN stimulation (5 or 10 Hz), inspiratory-modulated neurons distinctly lacked the orthodromic action potentials observed in ELM (Fig. 4). Decreased membrane depolarization and firing frequency in the neurons mirrored the diminished amplitude or absences in inspiratoryrelated phrenic nerve discharges.

3.

Fig. 3 – Spontaneous ELM burst. Intracellular recording reveals that the ELM burst is clearly separated from its post-inspiratory depolarization by the preceding small hyperpolarization (dot). Note that the hyperpolarization occurs between the normal inspiratory-related phrenic nerve discharge and the swallow-breath (asterisk).

2.3. Inspiratory-modulated neuron behaviour during SLN stimulation In addition to ELM, 3 inspiratory-modulated neurons were recorded approximately 300 μm ventral to the strong antidromic field potential produced by RLN stimulation. These

Table 1 – Characteristics of ELM bursts during SLN stimulation. Bursts from all ELM were pooled on the basis of their appearance during 5 or 10 Hz SLN stimulation. No significant differences were observed. SLN stimulation ELM burst

5 Hz

10 Hz

Number of bursts Mean frequency (Hz) Mean duration (ms) Mean depolarization (mV) Mean preceding hyperpolarization (mV)

10 81.2 ± 17.1 220.3 ± 89.7 6.6 ± 0.8 6.9 ± 2.0

17 87.2 ± 19.4 155.3 ± 38.9 11.6 ± 3.9 5.99 ± 3.0

p value n/a 0.42 0.31 0.06 0.43

Discussion

Experimental studies in swallow using animals have traditionally used cats, goats, sheep and dogs (Doty and Bosma, 1956; Feroah et al., 2002; Miller, 1972; Zoungrana et al., 1997). We previously reported that train stimulation of the SLN in the in vivo adult rat produces swallow-related bursting activity in ELM using extracellular recording (Sun et al., 2011b). Here, we extend our previous observations by examining changes in ELM membrane potential during SLN stimulation using intracellular recording. The main findings are, similar to ELM in cat (Gestreau et al., 2000; Shiba et al., 1999), ELM bursts elicited during SLN stimulation (and spontaneously after stimulation) are associated with a plateaulike membrane depolarization and are preceded by a small hyperpolarization in the anaesthetised, in vivo adult rat. Furthermore, our results from spontaneous ELM bursts suggest that this burst activity is not merely an extension of its post-inspiratory activity.

3.1.

Technical limitations

We believe that the burst activity observed in ELM is associated with swallow, although we did not monitor activity in nerves supplying swallow-related muscles (e.g. pharyngeal branches of the vagus nerve or the hypoglossal nerve). Swallow is the most frequently observed reflexive behaviour elicited by electrical stimulation of the SLN in rat (Kessler and Jean, 1985) , although high frequency SLN stimulation can elicit other protective upper airway behaviours such as cough and laryngeal adductor response in other species (Ambalavanar et al., 2004; Gestreau et al., 2000). Unlike animals such as sheep, cat or ferret, rats do not vomit or cough. Tussigenic stimuli such as intra-laryngeal capsaicin and electrical stimulation of the SLN in rat do not produce the stereotypical cough-related changes in phrenic and abdominal nerves observed during this behaviour in species such as cat (Ohi et al., 2004). Nevertheless, the membrane potential

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Fig. 4 – Responses in an inspiratory-modulated neuron in the vicinity of ELM. Inspiratory-related firing activity in the neuron gradually weakened in parallel to the decrease in phrenic nerve discharge amplitude with ongoing stimulation. The neuron could not be antidromically excited by ipsilateral RLN stimulation and therefore likely belong to the rostral ventral respiratory group (rVRG). The membrane potential of the neuron during the absence of an expected inspiratory-related phrenic nerve discharge (arrow) corresponds with the level it exhibited during post-inspiration.

changes recorded from ELM, and phrenic nerve activity, closely resemble that seen during swallow in cat (Shiba et al., 1999).

3.2. in rat

Changes in ELM membrane potential during swallow

During swallow, a temporally coordinated wave of contraction of muscles in the upper airway and upper alimentary tract propels food towards the stomach (Jean, 2001; Miller, 1982). Laryngeal adduction of the vocal cords by contraction of the thyroarytenoid, interarytenoid and cricothyroid muscles during the oropharyngeal phase of swallow normally prevents aspiration of the food bolus into the lower airways (Doty and Bosma, 1956; McCulloch et al., 1996). Correspondingly, intracellular recordings of hypoglossal (Roda et al., 2002; Tomomune and Takata, 1988), laryngeal (Gestreau et al., 2000; Shiba et al., 1999; Suzuki et al., 2010) as well as pharyngeal and esophageal (Zoungrana et al., 1997) motoneurons, in cat and sheep reveal that their swallow-related depolarization and firing activity is often associated with a hyperpolarization. The swallow-related hyperpolarization/ depolarization sequence is also observed in inspiratorymodulated cricothyroid motoneurons, whose axons run in the SLN, in rat (Saito et al., 2002a) and cat (Numasawa et al., 2004). The sequential inhibition then excitation of motoneurons during swallow is considered to enable the recruitment of each muscle at the appropriate time. Importantly, subtle differences are found in laryngeal adductor function during swallow in different species. In monkeys, the

thyroarytenoid muscle contracts 30–80 ms following the contraction of oropharyngeal muscles; in dogs and cats, this latency is 150–200 ms (Doty and Bosma, 1956). In cat, the cricothyroid muscle exhibits an initial inhibition followed by a contraction during swallow, while in dogs swallow-related inhibition often predominates. In addition, all cricothyroid motoneurons exhibit a hyperpolarization/depolarization in rat (Saito et al., 2002a). In contrast, some cricothyroid motoneurons recorded in cat show hyperpolarization or depolarization alone during swallow (Numasawa et al., 2004). Our study is the first demonstration in rat of a swallow-related hyperpolarization/depolarization sequence in ELM, whose axons run in the RLN and innervate the thyroarytenoid, interarytenoid and lateral cricoarytenoid muscles. The characteristics of swallow-related activity in ELM in rat are qualitatively and quantitatively very similar to those documented in ELM of cat, including mean depolarization (~10 mV), duration of depolarization (200 ms) and frequency of action potentials (close to 100 Hz) (Gestreau et al., 2000; Shiba et al., 1999). In cat, the hyperpolarization in ELM occurs during early swallow, coincident with the onset of the swallowrelated burst in hypogossal nerve that innervates tongue muscle (Shiba et al., 1999). The following depolarization in ELM occurs during the swallow-related hypogossal nerve burst. The source of swallow-related hyperpolarization of laryngeal motoneurons is unknown. Previous studies using chloride injection into intracellularly recorded ELM in cat (Gestreau et al., 2000), and cricothyroid motoneurons in rat (Saito et al., 2002a) and cat (Numasawa et al., 2004), demonstrate that it derives from chloride-mediated IPSPs. A recent

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investigation in cat by Suzuki et al. (2010) reported that the hyperpolarization is GABA-receptor mediated as its magnitude is attenuated by juxtacellular micro-iontophoresis of bicuculline, but not the glycine receptor antagonist strychnine. In cat, expiratory-augmenting neurons of the Bötzinger complex that monosynaptically inhibit and shape ELM activity during cough and sneeze, are not activated and therefore unlikely to be a source of inhibitory pre-motor input to ELM during swallow (Shiba et al., 2007). According to the model of the swallow central pattern generator proposed by Jean (2001), interneurons in the medullary reticular formation (dorsomedial to the rostral nucleus ambiguus) may be involved in the temporal coordination of UA muscle contraction since they have known axon collaterals to different UA motoneurons (Amri et al., 1990; Ezure et al., 1993; Sugiyama et al., 2011). Other candidate inhibitory pre-motoneurons include neurons in Roller's nucleus, which are shown to inhibit hypoglossal motoneurons during swallow (Ono et al., 1998). We are unable to comment further on the source of hyperpolarization in ELM as our experiments did not directly address this issue. It is unclear whether swallow-related ELM burst activity derives from the same mechanism underlying its normal post-inspiratory activity. The generation of post-inspiratory activity in ELM is still subject to debate (Bautista et al., 2010) but may derive from direct excitatory inputs from neurons in the Kölliker–Fuse nucleus in the dorsolateral pons (Dutschmann and Herbert, 2006). Interestingly, a recent study in awake goat demonstrated that chronic lesion of the parabrachial-Kölliker–Fuse region interferes with the coordination of breathing and swallowing, but not the contraction of laryngeal adductor muscle during the oropharyngeal phase (Bonis et al., 2011). In our earlier study, we reported a distinct ‘gap’ between post-inspiratory firing in ELM and non-respiratory-related burst activity using extracellular recording (Sun et al., 2011b). The occurrence of the two activities decreased and increased, respectively, with higher intensities of SLN stimulation. Here, our demonstration of an intervening swallow-related hyperpolarization, and the declining membrane potential during post-inspiration that precedes it, suggest that the plateau-like ELM depolarization is not a mere extension of the post-inspiratory depolarization. Interestingly, the EPSPs associated with action potentials comprising ELM bursts imply that the bursting activity requires an excitatory mechanism (either direct excitation or disinhibition). It is unlikely that ELM excitation and burst firing during swallow involve the Bötzinger complex as we have previously demonstrated that bursting activity in the RLN (as a surrogate for ELM bursts) is unaffected by reversible chemical inhibition of this area (Sun et al., 2011b). The current study confirms our previous finding that SLN stimulation further elicits orthodromic action potentials in ELM in addition to burst activity. The latency of orthodromic action potentials (~6 ms) was fairly consistent despite the frequency of SLN stimulation used. In earlier studies, similar orthodromic activities (sometimes referred to as ‘initial responses’) are described in other swallow-related motoneurons (Grélot et al., 1989; Zoungrana et al., 1997) and medullary interneurons (Ezure et al., 1993; Kessler and Jean, 1985; Saito et al., 2002b). The relatively short latency of orthodromic action potentials and the high fidelity of their occurrence in

ELM imply they arrive via an oligosynaptic pathway from the termination of SLN afferent fibers in the nucleus of the solitary tract (Bellingham and Lipski, 1992; Kalia and Mesulam, 1980). The significance of the orthodromic action potential is unknown, but may be related to the short latency contraction of laryngeal adductor muscle (without respiratory disturbances; termed the ‘laryngeal adductor response’) during mild laryngeal irritation (Ambalavanar et al., 2004; Sun et al., 2011a). The latency of the orthodromic action potential (~6 ms) is in good register with the latency of the onset of contraction of the thyroarytenoid muscle (~10 ms) during very low (0.5 Hz) SLN stimulation in cat (Ambalavanar et al., 2004). Tonic ELM firing, consisting of numerous orthodromic action potentials and occasional bursts, during high frequency train stimulation of the SLN may represent spasm of laryngeal adductor muscles in response to an ongoing laryngeal irritant, such as the insertion of endotracheal tubes (Rex, 1971).

3.3. in rat

Changes in rVRG membrane potential during swallow

We obtained additional recordings from inspiratorymodulated neurons that were located ventral to the loose formation of the nucleus ambiguus, which likely belonged to the rostral ventral respiratory group (rVRG). This region is a principal source of monosynaptic, excitatory input to phrenic motoneurons in rat (Dobbins and Feldman, 1994). Although we did not confirm the bulbospinal projection of these neurons, our results indicate they are probably phrenic premotoneurons since they showed attenuated depolarization and firing activity that mirrored changes in amplitude of inspiratory-related phrenic nerve discharges during SLN stimulation. Similarly, a portion of inspiratory-augmenting neurons in the ventral respiratory group are shown to be inhibited during swallow in cat (Oku et al., 1994). The authors also observed that some inspiratory-augmenting neurons are excited during fictive swallowing; however, none such neurons were encountered in this study. No short latency, orthodromic action potentials were observed in the inspiratorymodulated neurons, consistent with the inhibition of phrenic nerve discharge during SLN stimulation.

3.4.

Swallow-related changes in breathing

Some differences in breathing during swallow have been noted between cat and rat. In cat, ‘phrenic breakthroughs’ (also called ‘swallow breaths’) refer to very small amplitude and short duration phrenic discharges that are elicited simultaneously with swallows during train SLN stimulation and are not respiratory related (Jodkowski and Berger, 1988; Grélot et al., 1992; Oku et al., 1994). Swallow breaths appear to be of very short duration (Oku et al., 1994). In this species, swallow breaths represent short lasting activation of phrenic motoneurons originating from bursts of activity in inspiratorymodulated pre-motoneurons in the rVRG and dorsal respiratory group (Grélot et al., 1992; Gestreau et al., 1996; Oku et al., 1994). Evidence in humans suggests swallow-related diaphragmatic activity does not produce airflow (Hårdemark Cedborg et al., 2009); thus, the function of paradoxical phrenic

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activity during swallow remains unknown. In our study, we also observed small amplitude, swallow-related phrenic discharges during SLN stimulation that occur coincident with ELM bursts. These discharges appear to be longer in duration compared with phrenic breakthroughs in cats (>0.3 s vs. <0.3 s in Oku et al. (1994). This is also seen, but not discussed, in a previous study in rat by Saito et al. (2002a,b). Although we did not record from inspiratory-augmenting neurons in the rVRG during swallow breaths (see above), the origin of such activity in rats likely originates from these neurons given they constitute the bulk of excitatory phrenic premotoneurons in this species (Dobbins and Feldman, 1994). Here, ‘swallow breaths’ also refer to swallow-related phrenic activity during spontaneous swallows following termination of SLN stimulation. These swallow breaths are similar to the small amplitude phrenic discharges during SLN stimulation in that they always occur with ELM bursts, and in the post-inspiratory period of phrenic discharge. However, they are noticeably greater in amplitude than those elicited during SLN stimulation (but still much reduced compared to inspiratory-related discharges). Very similar phrenic activity in the P-I phase was previously reported in association with spontaneous swallows after SLN stimulation in rat, by Saito et al. (2002a,b). To our knowledge, such activity during spontaneous swallows in cat has not been investigated.

3.5.

Conclusion

It is important to establish similarities (or differences) in laryngeal adductor behaviour in rat compared to other species that are capable of elaborating more complex oropharyngeal behaviours, including cough and vomiting. SLN train stimulation in rat elicits a hyperpolarization/depolarization and subsequent bursting activity in ELM, which is similar to the behaviour of ELM in cat during swallow. Therefore, the in vivo rat preparation may be used to study swallow-related mechanisms at the level of the larynx given the increasing use of rodent preparations in studies of the central control of respiration and its coordination with other functions (e.g. cardio-respiratory coupling). It remains to be determined if there are underlying biophysical properties of rat ELM that distinguish them from ELM in other species (e.g. Shiba et al., 1999).

4.

Experimental procedures

This study was approved by the Macquarie University Animal Ethics Committee. All experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals as endorsed by the National Health and Medical Research Council of Australia.

Briefly, the femoral artery and vein were cannulated for measurement of arterial blood pressure and drug/fluid administration, respectively. The animal was tracheostomised below the level of the larynx to permit mechanical ventilation. The left phrenic nerve was dissected for recording while the ipsilateral SLN and RLN were dissected for stimulation. Following nerve dissections, the animal was mounted on a stereotaxic frame, its lumbar spine clamped and head flexed roughly 45° such that its spinal cord and medulla were taut and horizontal. Mechanical ventilation was started and neuromuscular blockade established (pancuronium bromide; i.v. 1 mg/kg and 0.5 mg/kg/h thereafter). Nerves were mounted on bipolar silver electrodes and kept moist by paraffin oil. Since the animals were not vagotomised, frequency of phrenic nerve discharge was entrained to the ventilation frequency in a 1:1 ratio as previously reported (Sun et al., 2011b). A laminectomy (C3–T1) was performed to facilitate minimal movement of the brainstem during intracellular recording. The dorsal surface of the medulla was exposed by a wide occipital craniotomy. The animal was maintained at a rectal temperature of 36–37 °C by means of a homeothermic blanket and heating lamp. 4.2.

Experimental protocol

The threshold stimulus intensity to elicit phrenic apnea for an entire 5 s of SLN train stimulation was determined at 20 Hz (0.2 ms pulse width) at increasing voltages starting at 1 V. The experimental intensity for SLN stimulation (regardless of frequency of stimulation) was 1–1.5× the threshold stimulus voltage. The caudal nucleus ambiguus (around the level of calamus scriptorius) containing the bulk of ELM was mapped by searching for antidromic field potentials following stimulation of the RLN (5–10 V, 1 Hz, 0.2 ms pulse width). During field mapping, a low impedance borosilicate glass electrode was used (1–5 MΩ, 3 M KCl, Harvard Apparatus, Dover, MA). Intracellular recordings of ELM were obtained using high impedance borosilicate electrodes (30–60 MΩ, 3 M KCl). Impaled neurons were confirmed as ELM on the basis of antidromic stimulation from the RLN, a positive collision test and the exhibition of the characteristic post-inspiratory modulation of membrane potential. Neurons that displayed a stable, average membrane potential more negative than −35 mV were included in this study. Following impalement of ELM, 5 s SLN stimulation trains (5, 10 or 20 Hz) were applied. Typically, the lowest frequency SLN stimulation was applied first. Stimulation trains were interspersed with 30 s recovery periods. At the conclusion of experiments, animals were euthanized with a 0.5–1 ml bolus i.v. injection of 10% KCl. 4.3.

4.1.

59

Data acquisition

Animal preparation

Six adult male Sprague–Dawley rats (400–600 g; Animal Resource Centre, WA, Australia) were deeply anaesthetised with an intraperitoneal injection of sodium pentobarbital (72 mg/ kg) and surgical anaesthesia was maintained throughout the experiment by additional intravenous administration of the same anaesthetic as required (~3 mg/kg/h).

All physiological data were acquired using the Power 1401plus ADC (Cambridge Electronic Design, Cambridge, UK) and recorded with the supplied Spike2 (v.6 or 7) program. Phrenic nerve activity (PNA) was pre-amplified (10×), then amplified (×2000), band-pass filtered (0.1–3 kHz) (CWE, Ardmore PA) and sampled at 3 kHz. DC recordings of intracellularly recorded neurons were amplified 10× and sampled at 10,000 Hz. Nerve and

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single cell recordings were additionally passed through a humbug device (Quest Scientific, Canada) prior to sampling to remove ambient 50 Hz electrical noise. 4.4.

Data analysis

Data analysis was performed offline using the Spike2 (v.7) program. The stimulus artefact during SLN stimulation was removed during analysis of ELM bursts by creating a ‘Wavemark’ channel in which only recorded action potentials were displayed. ELM bursts were identified using the ‘burst’ script. Both the longest inter-spike interval within a burst and the maximum inter-burst interval were specified as 5 ms. Minimum number of spikes constituting a burst was 5. The mean frequency and duration of ELM bursts were calculated using the burst analysis script. The mean depolarization during each identified ELM burst was determined by calculating the difference between this average membrane potential during the burst, and the membrane potential during the inspiratory hyperpolarization of the last phrenic nerve discharge prior to the SLN stimulus train. The magnitude of the hyperpolarization associated with ELM bursts was determined by calculating the difference between the highest membrane potential just before, and the nadir during, the hyperpolarization. For each neuron, ELM bursts during stimulus trains were summed and averaged for each frequency of SLN stimulation. During analysis, ELM bursts from all neurons were grouped according to SLN stimulation frequency since data could not be obtained at the three intensities of SLN stimulation (5, 10 and 20 Hz) for all neurons. An unpaired t-test was used to compare characteristics of ELM bursts occurring during 5 Hz and 10 Hz SLN stimulation. All data is reported as mean ± SD. For image production, the phrenic neurogram was rectified and smoothed (τ = 0.01 s). Stimulus artefacts were reduced in DC recordings of ELM neurons.

Acknowledgments The authors’ work is funded by Macquarie University, the ARC (DP110102110), NHMRC (457080, 604002, 1024489, and 1030297) and a Macquarie University Research Excellence Scholarship to TGB.

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