Neuroscience Research 80 (2014) 17–31

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Activity of respiratory neurons in the rostral medulla during vocalization, swallowing, and coughing in guinea pigs Yoichiro Sugiyama a,∗ , Keisuke Shiba b , Shigeyuki Mukudai a , Toshiro Umezaki c , Yasuo Hisa a a

Department of Otolaryngology-Head and Neck Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan Hikifune Otolaryngology Clinic, Sumida, Tokyo 131-0046, Japan c Department of Otolaryngology, Graduate School of Medicine, Kyushu University, Fukuoka 812-8582, Japan b

a r t i c l e

i n f o

Article history: Received 23 October 2013 Received in revised form 4 December 2013 Accepted 18 December 2013 Available online 29 December 2013 Keywords: Respiration Vocalization Swallowing Coughing Guinea pigs

a b s t r a c t To examine the relationship between the neuronal networks underlying respiration and non-respiratory behaviors such as vocalization and airway defensive reflexes, we compared the activity of respiratory neurons in the ventrolateral medulla during breathing with that during non-respiratory behaviors including vocalization, swallowing, and coughing in guinea pigs. During fictive vocalization the activity of augmenting expiratory neurons ceased, whereas the other types of expiratory neurons did not show a consistent tendency of increasing or decreasing activity. All inspiratory neurons discharged in synchrony with the phrenic nerve activity. Most of the phase-spanning neurons were activated throughout the vocal phase. During fictive swallowing, many expiratory and inspiratory neurons were silent, whereas many phase-spanning neurons were activated. During fictive coughing, many expiratory neurons were activated during the expiratory phase of coughing. Most inspiratory neurons discharged in parallel with the phrenic nerve activity during coughing. Many phase-spanning neurons were activated during the expiratory phase of coughing. These findings indicate that the medullary respiratory neurons help shape respiratory muscle nerve activity not only during breathing but also during these non-respiratory behaviors, and thus suggest that at least some of the respiratory neurons are shared among the neuronal circuits underlying the generation of breathing and non-respiratory behaviors. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

1. Introduction The medullary respiratory neuronal networks generate respiratory rhythm and regulate the motoneurons of the respiratory and upper airway muscles to take in oxygen and release carbon dioxide in the lung. The organization of this neuronal circuitry consists of a longitudinal column extending from the facial nucleus to the rostralmost part of the cervical spinal cord in the lateral tegmental field; it is divided into several subregions including the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG), Bötzinger complex (BötC), preBötzinger complex (preBötC), the rostral ventral respiratory group (rVRG), and the caudal ventral respiratory group (cVRG) (Feldman and Del Negro, 2006). Interestingly, vocalization and the airway defensive reflexes such as swallowing and coughing are also generated by contractions of the same respiratory and upper airway muscles. However, the physiological

∗ Corresponding author at: Department of Otolaryngology-Head and Neck Surgery, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto City 602-8566, Japan. Tel.: +81 75 251 5603; fax: +81 75 251 5604. E-mail address: [email protected] (Y. Sugiyama).

roles of these non-respiratory behaviors are not gas exchange but rather vocal communication, feeding, and expelling a foreign body from the airway. The phenomenon that respiratory rhythm is modulated in synchrony with these non-respiratory behaviors strongly suggests a close relationship between the neuronal networks responsible for respiration and those for non-respiratory movements. Previous studies have proposed that networks of the neurons controlling breathing and those controlling these behaviors are likely shared (Funk and Feldman, 1995). To determine whether these neuronal networks overlap and whether the respiratory neurons are shared among them, we compared the activity of the respiratory neurons during breathing and during these nonrespiratory behaviors in guinea pigs in the present study. We focused on the respiratory neurons located between the BötC and rVRG, and recorded their extracellular activity during fictive behaviors in anesthetized paralyzed guinea pigs, which allow stable recording by eliminating animal movements and simplify the data analysis by removing movement-related feedback inputs. Fictive vocalization was evoked by electrical stimulation of the midbrain periaqueductal gray (PAG) or its descending pathway (Shiba et al., 1996; Sugiyama et al., 2010). Fictive swallowing was elicited by electrical stimulation of laryngeal afferents, i.e., superior laryngeal

0168-0102/$ – see front matter © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. http://dx.doi.org/10.1016/j.neures.2013.12.004

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Y. Sugiyama et al. / Neuroscience Research 80 (2014) 17–31

nerve (Nishino et al., 1985), and fictive coughing was evoked by mechanical stimulation of the trachea (Bolser, 1991).

A

Vocalization

2. Materials and methods

SLN

All experimental procedures conformed to the Physiological Society of Japan Principles for the Care and Use of Animals, and were approved by the University Committee for the Use of Animals in Research.

ABD PHR Call site stim

2.1. General surgical procedures Data were collected from 20 adult male guinea pigs (Hartley, 6–12 weeks, Shimizu Laboratory Supplies, Kyoto, Japan) weighing 350–600 g. Each animal was anesthetized with urethane (initially 0.7 g/kg, 0.5 g/kg as necessary, intraperitoneally), and a catheter was inserted into the common carotid artery to record blood pressure. The level of anesthesia was titrated to maintain blood pressure at ∼100 mm Hg and to prevent both spontaneous and reflexive movements. The trachea was intubated, and a femoral vein was cannulated for drug administration. The superior laryngeal nerve (SLN), recurrent laryngeal nerve (RLN), C5 phrenic nerve (PHR), and L1 abdominal nerve (ABD) were isolated bilaterally, secured in bipolar silver cuff electrodes that were insulated with silicone, and covered with a mixture of Vaseline and mineral oil. Dexamethasone (1 mg/kg) and atropine (0.1 mg/kg) were intramuscularly administered to minimize brain edema and to reduce secretions in the airways, respectively. Rectal temperature was maintained at 37–38 ◦ C using a DC-powered heating pad. The animals were positioned in a stereotaxic frame. An occipital craniotomy was performed to expose the caudal brainstem. The caudalmost part of the cerebellum was gently retracted and aspirated to permit the insertion of electrodes into the area of the medulla near the obex. The animal was then paralyzed using Vecuronium bromide (Fuji Pharma, Tokyo, Japan; initial injection of 0.3 mg/kg intravenously,

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Coughing

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Fig. 1. Motor patterns of fictive vocalization, swallowing, and coughing. Electrical stimulation of the periaqueductal gray or pontine call site evoked fictive vocalization. The vocal phase was identified by bursting activity of the superior laryngeal (SLN) and the abdominal nerve (ABD) followed by activation of the phrenic nerve (PHR) (A). Electrical stimulation of the SLN elicited fictive swallowing identified by bursting activity of the recurrent laryngeal nerve (RLN) (arrowhead) (B). Fictive coughing, which was evoked by mechanical stimulation of the trachea, consisted of an abrupt burst of the abdominal nerve accompanied by bursting activity of the RLN following phrenic nerve activation (C). Horizontal bars at the bottom of panels indicate the periods of electrical or mechanical stimulation that evoked fictive behaviors.

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Fig. 2. Subtypes of respiratory neurons in the rostral ventrolateral medulla. Expiratory neurons with an augmenting (E-AUG) (A), decrementing (E-DEC) (B), and constant (ECON) (C) firing patterns, exhibiting a gradual increase, decrease, and no change in firing rates during the expiratory phase, respectively. Inspiratory neurons with augmenting (I-AUG) (D), decrementing (I-DEC) (E), and constant (I-CON) (F) firing patterns. Panels G and H show cell firings with phase spanning activity which began during inspiration and continued into expiration (inspiration to expiration phase spanning: IE) and began during expiration and continued into inspiration (expiration to inspiration phase spanning: EI), respectively. Inst freq.; instantaneous frequency.

7 43 1 10 2 26 4 7

Number of respiratory neurons recorded during vocalization, swallowing, and coughing are indicated. (E) and (I) indicate that the activity was analyzed during the expiratory and inspiratory phases, respectively. “Increased” and “Decreased” indicate that the peak firing rates were increased and decreased compared to that of control respiration, respectively. Values in parentheses for expiratory and phase-spanning neurons indicate mean percent changes (± SEM) in the peak firing rate from control expiration, whereas inspiratory neurons indicate that from control inspiration.

4 22 4 8 0 2 0 1 2 15

16 (55 ± 17%) 2 (123 ± 145%) 1 (36%) 36 (158 ± 21%) 9 (158 ± 48%) 3 (175 ± 82%)

24 (157 ± 25%)

Activated (I) (% increase in firing rate) Activated (E) Total

13 (46 ± 11%)

2 18

0 14

18 (247 ± 42%) 4 (359 ± 116%) 14 (215 ± 42%)

Activated (E) (% increase in firing rate) Silent (E) Total

15 7 22 8 7 15 Activated Silent Total 5 17 22 1 2 3 0 4 4 4 11 15 Activated Silent Total 18 33 51 1 10 11 16 15 31 1 8 9

26 4 4 18 Total 14 52 4 12 1 31 9 9

20 (−39 ± 6%) 4 (−33 ± 9%) 16 (−40 ± 7%) 0

7 0 7

3 10 0 16

3 26

0 1 (−50%)

1 (−50%)

7 (191 ± 75%) 15 (96 ± 25%) 4 (93 ± 52%)

Vocalization Activated (E) Increased (% increase in firing rate) Decreased (% decrease in firing rate) Silent (E) Total Swallowing Activated Silent Total Coughing Activated (E) (% increase in firing rate) Silent (E) Total

0

14 (169 ± 86%)

18 (152 ± 67%)

Activated (I)

(% change in firing rate)

18

(−7 ± 7%)

4

(6 ± 15%)

4

(−6 ± 18%)

26

(−5 ± 6%)

Activated (E) Increased (% increase in firing rate) Decreased (% decrease in firing rate) Silent (E) Total

EI IE DEC AUG Total CON DEC AUG

Expiratory neurons

Table 1 Responses of respiratory neurons during fictive vocalization, swallowing, and coughing.

Inspiratory neurons

CON

Total

Phase-spanning neurons

Total

22 (127 ± 30%)

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maintained by hourly injections of 0.15 mg/kg) and artificially ventilated with room air (30–50 cycles/min). A bilateral pneumothorax was induced to reduce ventilation-related brain movements. Endtidal CO2 was maintained at 3–5%. An intravenous infusion of epinephrine in saline at the rate of 0.005–0.01 mg/kg/min was administered, when the mean blood pressure decreased below 80 mmHg. A constant level of anesthesia was maintained by injecting 0.5 g/kg doses of urethane whenever needed as judged by an increase in blood pressure (>100 mmHg) and instability of respiratory rhythm identified by the phrenic nerve activity. At the end of the experiment, animals were perfused transcardially with 300 mL of physiological saline followed by 300 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), under deep anesthesia (an additional dose of urethane was applied, if needed).

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Fig. 3. Activity of three expiratory neurons that fired with more intense (A, B) and less intense firing (C) during vocalization than during control respiration. A, B: These E-DEC and E-CON neurons in the rVRG showed increased firing rates during vocalization compared to before stimulation. C: The firing rate of this E-DEC neuron in the BötC was decreased during vocalization compared to during control expiration. When the call site stimulation was initiated during inspiration, the vocal-related SLN and ABD bursts were evoked subsequent to the phase switching from the inspiratory phase to the vocal phase (A, B). In contrast, the stimulation which started during expiration directly evoked the vocal activity without preceding phase transition (C). Vertical dashed lines indicate respiratory phase transitions judged from phrenic activity. The period of call site stimulation in each panel is indicated by a thick horizontal bar (stim).

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2.2. Electrophysiological procedures To evoke fictive vocalization, we delivered electrical stimulation (pulse duration, 0.2 ms; frequency, 100 Hz; intensity, 30–60 ␮A, 1.5 times threshold for evoking fictive vocalization) to the PAG or the pontine call site in the dorsal pontine tegmentum (De Lanerolle, 1990; Sugiyama et al., 2010) through the tip of a 1 M tungsten microelectrode (Unique Medical Co., Ltd., Tokyo, Japan). Fictive vocalization was confirmed by bursting activity of the SLN, which innervates the vocal tensor muscles and contributes to the pitch control during vocalization, and abdominal nerve as reported by Shiba et al. (1996). The electrode tip was positioned where electrical stimulation produced a series of bursting activities of the SLN and abdominal nerve, i.e. vocal phase, followed by phrenic nerve activation as shown in Fig. 1A. Fictive swallowing was elicited by electrical stimulation of the SLN (pulse duration, 0.2 ms; frequency, 10–20 Hz, intensity 50–80 ␮A), and identified by bursting activity of the RLN as shown in Fig. 1B (Nishino et al., 1985; Sugiyama et al., 2011). Fictive coughing was evoked by mechanical irritation of tracheal mucosa using a silicon tube inserted through the tracheostoma, or by electrical stimulation of the RLN which contains the afferent fibers of the trachea and larynx (pulse duration, 0.2 ms; frequency, 10 Hz, intensity 40–60 ␮A), and identified by the bursting activity of RLN and the abdominal nerve preceded by increased and prolonged phrenic nerve activity as shown in Fig. 1C (Bolser, 1991; Grélot and Milano, 1991). Activities of medullary respiratory neurons were recorded extracellularly with a glass micropipette filled with 3 M KCl (tip impedance 5–10 M). Penetration was from 0.0 to 4.0 mm rostral to the obex, and from 1.5 to 2.5 mm lateral to the midline. The electrode tip was positioned where the respiratory-modulated discharge of a single cell was recorded, and then the call site was stimulated to evoke fictive vocalization. If the recorded cell was antidromically activated from the RLN or SLN, the cell was not recorded in the present study. Since some units were not tested for responses to the antidromic stimulation of the RLN or SLN, these neurons possibly included the laryngeal motoneurons. An electrolytic lesion was made at the stimulating call site by passage of a 100-␮A negative current for 30–60 s through a tungsten microelectrode. Some recording locations were also marked by electrolytic lesions through the recording electrode. Unit activity was amplified (MEZ-8301, Nihon Kohden, Tokyo, Japan) and sampled at 20,000 Hz using a Power 1401 mk 2 data collection system and Spike2 version 6 software (Cambridge Electronic Design, Cambridge, UK). Activity recorded from each nerve was amplified by a factor of 10,000, filtered with a band pass of 100–10,000 Hz, integrated with a 1-ms time constant, full-wave rectified, and sampled at 2000 Hz. For all data obtained, the spike detection and sorting feature of the Spike2 software was used to delineate the occurrence of neuronal firing. The peak firing rates of recorded cells during breathing were compared with those during vocalization and those during coughing. The peak firing rates were calculated by averaging the occurrence of spikes within 0.2 s during which the instantaneous frequency reached the maximum during each respiratory cycle. Stimulus artifacts during call site stimulation at 0.5–0.9 ms after each repetitive stimulus were eliminated, and thus discharges were not counted during this period of artifact cancelation.

digital camera (DP21, Olympus) and montages of images were assembled using PTGui-Pro photo-stitching software (New House Internet Services BV, The Netherlands). Adobe Illustrator software (Adobe systems, San Jose, CA) was used for drawings of the sections. Recording sites were reconstructed on these drawings with reference to the locations of electrical lesions, the relative positions of electrode tracks, and microelectrode depths. Pooled data are represented as mean ± one standard error. Statistical significance was set at P < 0.05. 3. Results Three types of respiratory neurons were recorded in the ventrolateral medulla: expiratory, inspiratory, and phase-spanning neurons. The expiratory and inspiratory neurons were subdivided into the following subtypes according to their discharge patterns: expiratory and inspiratory neurons with augmenting (E-AUG and I-AUG) (Fig. 2A and D), decrementing (E-DEC and I-DEC)

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2.3. Histological procedures and statistical analyses After tissue fixation, the brainstem was cut transversely at 50 ␮m using a freezing microtome. Tissue sections were stained using cresyl violet. Brainstem sections were observed using an upright microscope (BX51, Olympus, Tokyo, Japan) with a CCD

Fig. 4. Vocalization-inactive expiratory neurons. An E-AUG neuron in A and an ECON neuron in C were silent during the period of SLN and ABD bursts corresponding to the vocal phase (V in C), but fired at the end of the stimulus-induced expiration during which the bursts were attenuated. An E-DEC neuron in between the rostral portion of rVRG and the BötC in B was silent throughout the expiratory phase when the bursts persisted to the end of expiration without attenuation.

Y. Sugiyama et al. / Neuroscience Research 80 (2014) 17–31

(Fig. 2B and E), and constant firing patterns (E-CON and I-CON) (Fig. 2C and F). The phase-spanning neurons were subdivided into inspiratory–expiratory (IE) (Fig. 2G) and expiratory-inspiratory (EI) (Fig. 2H) neurons. The peak firing rates of E-AUG, E-DEC, and E-CON neurons were 36.6 ± 5.5, 30.9 ± 2.5, and 41.0 ± 6.8 Hz, respectively. I-AUG, I-DEC, and I-CON neurons discharged with a maximum frequency of 69.0 ± 11.2, 50.0 ± 5.9, and 43.3 ± 13.0 Hz, respectively. The peak firing rates of IE and EI neurons during the expiratory phase were 25.9 ± 3.3 and 13.1 ± 3.6 Hz, respectively. A total of 104 respiratory neurons were recorded during fictive breathing, vocalization, swallowing, or coughing (9 E-AUG, 31 E-DEC, 12 E-CON, 18 I-AUG, 4 I-DEC, 4 I-CON, 16 IE, and 10 EI) (Table 1), of which 77 neurons were tested for responses to all these behaviors

21

(7 E-AUG, 26 E-DEC, 9 E-CON, 14 I-AUG, 1 I-DEC, 2 I-CON, 13 IE, and 5 EI). 3.1. Vocalization We recorded the activity of 52 expiratory, 26 inspiratory, and 26 phase-spanning neurons during fictive vocalization. Call site stimulation induced various responses in expiratory neurons. Approximately 75% of recorded expiratory neurons were activated (Fig. 3) and the others were silent during the vocal phase (Fig. 4). The former cells were classified into two categories according to their firing rates: the first (35% of the whole population) (Fig. 3A and B) and second (38%) (Fig. 3C) category cells showed

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Fig. 5. Firing of I-AUG neurons during call site stimulation. (A, B) These I-AUG neurons exhibited the same discharge patterns as those of the phrenic nerve during stimulation: the plateau-like discharge pattern was observed when the phrenic nerve exhibited the bell-shaped or plateau-like phrenic activity (A), while the augmenting pattern was observed when the phrenic nerve exhibited the augmenting phrenic activity (B). (C) This I-AUG neuron fired only at the end of inspiration during control breathing, but it fired throughout the inspiratory phase and exhibited an augmenting discharge pattern during stimulation. The time axis of each box in the left panel is expanded in the right box. The second trace in B (I-AUG) was truncated because of saturation in spike amplification.

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increased and decreased peak firing rates, respectively, during the vocal phase compared to during normal breathing (Table 1). In all these active cells, call site stimulation evoked a continuous or bell-shaped firing pattern during the vocal phase (Fig. 3). While a vast majority of the E-DEC neurons were activated during the vocal phase, all E-AUG and some E-CON neurons were silent during the vocal phase. The vocal-related activity identified by the SLN and abdominal nerve bursts generally continued throughout the expiratory phase (e.g. Fig. 3B). This vocal activity sometimes terminated or attenuated at the end of expiratory phase (e.g. Fig. 4C), during which more than half of the silent cells, 5 E-AUG neurons and 3 E-CON neurons, discharged (Fig. 4A and C). All recorded inspiratory neurons were activated during the inspiratory phase during call site stimulation, and their activities were synchronized to the stimulus-induced phrenic nerve activity (Figs. 5 and 6). The augmenting firing pattern of the phrenic activity was usually changed to a bell-shaped or plateau-like pattern during stimulus-induced inspiration (Figs. 5A, C, and 6), but sometimes it remained as an augmenting pattern (Fig. 5B). Most I-AUG neurons exhibited the same firing pattern as the phrenic response: the augmenting firing pattern was altered to continuous when the phrenic nerve showed a bell-shaped or plateau-like pattern during stimulation (Fig. 5A) or it remained as an augmenting pattern when the phrenic discharge pattern remained unchanged during stimulation (Fig. 5B). Four I-AUG neurons that discharged only at the end of control inspiration exhibited the augmenting firing pattern even when the phrenic nerve exhibited a bell-shaped

or plateau-like pattern (Fig. 5C). In all recorded 4 I-DEC and 4 I-CON neurons, the discharge patterns remained unchanged during stimulation (Fig. 6). We recorded 16 IE and 10 EI neurons during the call site stimulation. All IE neurons fired during the vocal phase. Their firing rates during vocalization were higher than those during control breathing in all but one cell (Fig. 7A). Seven of 10 EI neurons fired during the vocal phase with more-intense firing rates compared to control expiration (Fig. 7B, Table 1). On the contrary, 3 of 10 EI neurons ceased to discharge during the vocal phase and fired only during stimulus-induced inspiration. Fig. 8 indicates the locations of the respiratory neurons recorded during fictive vocalization in the rostral ventrolateral medulla. The neurons were located 0.05–3.75 mm rostral to the obex, 1.5–2.35 mm lateral to the midline, and 1.6–3.1 mm depth from the dorsal surface. E-AUG neurons were recorded in the rostral parts of respiratory neuron columns (2.53 ± 0.23 mm rostral to the obex) corresponding to the area between the BötC and rostral portion of the rVRG. Inspiratory neurons were located in the rVRG not in the BötC, meanwhile E-DEC, E-CON, and phase-spanning neurons were broadly distributed throughout the columns. The expiratory neurons whose firing rate increased during the vocal phase were distributed more caudally (1.43 ± 0.22 mm rostral to the obex) compared to those with a decreased activity (2.23 ± 0.18 mm rostral to the obex) and those that were silent (2.53 ± 0.15 mm rostral to the obex); Dunn’s multiple comparison test was applied after Kruskal–Wallis test (nonparametric one-way ANOVA, P = 0.0023). In addition, E-DEC neurons with an increase in firing rate during vocalization were located more caudally (1.61 ± 0.25 mm rostral

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Fig. 6. Examples of firing patterns of I-DEC and I-CON neurons during call site stimulation. The I-DEC neuron (A) showed a decrementing firing pattern, whereas the I-CON neuron (B) exhibited a continuous firing pattern during stimulation. The data in Figs. 9E and 12C were obtained from this I-CON neuron. The time axis of each box in the left panel is expanded in the right box.

Y. Sugiyama et al. / Neuroscience Research 80 (2014) 17–31

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that were activated during swallowing and those that were silent were intermixed in the rostral ventrolateral medulla.

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Fig. 7. Activity of phase-spanning neurons during vocalization. (A) An IE neuron strongly fired during the vocal phase. This neuron sometimes ceased its firing when the vocal-related SLN and ABD bursts were attenuated at the end of the expiratory phase during the call site stimulation. (B) An EI neuron weakly fired during the late expiration of control respiration, but strongly fired throughout the vocal phase. The second trace in B (EI) was truncated because of saturation in spike amplification.

to the obex) than those whose activities decreased or ceased (2.19 ± 0.20 mm rostral to the obex), although these were not shown to be significantly different using the Mann–Whitney test. 3.2. Swallowing We observed swallow-related activity of 95 respiratory neurons including 51 expiratory, 22 inspiratory, and 22 phase-spanning neurons, which were also tested for responses to the call site stimulation. Fig. 9 illustrates the responses of each type of neuron to fictive swallowing. Of 51 expiratory neurons, 33 cells were silent, whereas the other 18 cells discharged at a frequency of 38.1 ± 11.0 Hz during swallowing. A majority of the E-AUG (89%) and E-CON (91%) neurons were silent during swallowing (Fig. 9A and B), whereas half of the E-DEC neurons (52%) were activated (Fig. 9C). Of 22 inspiratory neurons, 17 cells (nearly 75% of I-AUG and I-CON, and all I-DEC neurons) were silent, whereas the other 5 cells (4 I-AUG and 1 I-CON) fired at 24.3 ± 9.3 Hz during swallowing (Fig. 9D and E). Two I-AUG neurons were activated during swallow-breath identified by the weak phrenic nerve activity during swallowing (Fig. 9D) (Dick et al., 1993). One swallowing-active I-AUG neuron fired only at the end of the swallow-related RLN burst. Fifteen of 22 phase-spanning neurons discharged during swallowing at a frequency of 18.8 ± 4.9 Hz. Eight IE neurons were activated (Fig. 9F), whereas 7 were silent (Fig. 9G) during swallowing. All recorded EI neurons discharged during swallowing, of which 3 neurons fired after the peak of the RLN burst (Fig. 9H). The locations of these respiratory neurons whose activity was analyzed during swallowing are indicated in Fig. 10. The expiratory neurons

We recorded the activity of 83 respiratory neurons during fictive coughing including 43 expiratory, 18 inspiratory, and 22 phasespanning neurons, whose vocal-related activity was also analyzed. Figs. 11–13 show examples of the firing pattern for each unit type. Of the 43 expiratory neurons, 36 neurons were activated during the abdominal burst corresponding to the expiratory phase of coughing, whereas 7 neurons were silent. The cough-related activity of E-AUG neurons exhibited various patterns: 3 neurons were strongly activated during the abdominal burst (Fig. 11A), and the other 4 were silent (Fig. 11B). The cough-related RLN burst generally persisted throughout the abdominal burst. However, in only 3 guinea pigs coughing events ceased the RLN burst during the second half of the abdominal burst (Fig. 11A). In such situations, 2 EAUG neurons were silent during the RLN burst and then discharged strongly for the remainder of the abdominal burst (Fig. 11A). Most E-DEC (24/26) and E-CON (9/10) neurons discharged during the cough-related abdominal bursts (Fig. 11C). Thirteen of 15 I-AUG neurons fired with an augmenting pattern during the inspiratory phase of coughing (Fig. 12A). All of the I-DEC and I-CON neurons exhibited a decrementing and a plateau-like activity during the inspiratory phase of coughing, respectively (Fig. 12B and C). Meanwhile, 2 I-AUG neurons that discharged only at the end of control inspiration were activated during the expiratory phase of coughing (Fig. 12D). All of the IE neurons were activated during the expiratory phase of coughing; their discharge rate was maximum at the beginning of the expiratory phase of coughing (Fig. 13A). Four EI neurons exhibited a decrementing activity during the expiratory phase of coughing (Fig. 13B), whereas the other 4 neurons were silent (Fig. 13C). Fig. 14 shows the location of the respiratory neurons whose activity was recorded during coughing. The expiratory neurons with the different response types were intermingled in the rostral ventrolateral medulla. 4. Discussion The present study showed that the respiratory neurons in the rostral ventrolateral medulla exhibited both varied and specific responses during vocalization and the airway reflexes including swallowing and coughing. All of the recorded respiratory neurons changed their activity in synchrony with these behaviors. We recorded the activity of some respiratory neurons during these behaviors with specific firing patterns which have not been previously reported. This evidence suggests that the brainstem respiratory neuronal networks help shape the motor commands of these behaviors, and supports the notion that the medullary respiratory neurons are multifunctional and can be shared in the motor neuronal circuits underlying these non-respiratory behaviors. 4.1. Vocalization Neurons sampled from the BötC were mainly E-AUG and EDEC neurons, whereas various types of respiratory neurons were collected from the rVRG. All of the recorded E-AUG neurons in the BötC were silent during vocalization, whereas the E-DEC neurons exhibited various firing patterns as demonstrated in previous studies (Sakamoto et al., 1996; Nonaka et al., 1999). Many E-DEC neurons in the rVRG fired during vocalization, some of which are possibly upper airway respiratory motoneurons including laryngeal motoneurons (Ezure, 1990; Zheng et al., 1991; Bianchi et al., 1995). On the other hand, the expiratory neurons in the BötC including E-AUG and E-DEC neurons are a

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A (mm) 4

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Fig. 8. Locations of respiratory neurons in the rostral ventrolateral medulla tested for responses to stimulation to the call site. Locations of cells are plotted on horizontal (A) and transverse sections (B–F). Levels indicated as panels B–F in the horizontal plane correspond to panels B–F in the transverse plane. Filled circles, filled triangles, and filled squares indicate E-AUG, E-DEC, and E-CON neurons, whereas open circles, open triangles, and open squares designate I-AUG, I-DEC, and I-CON neurons, respectively. Filled and open stars indicate IE and EI neurons, respectively. For expiratory and phase-spanning neurons, red and gray symbols indicate neurons with more-intense and less-intense firing during vocalization than during control expiration, respectively, whereas black symbols designate neurons that were silent during the vocal phase. Numbers at the upper right corner of each panel in B–F indicate the distance (in mm) separating the brainstem sections from the obex. AP, area postrema; DMV, dorsal motor nucleus of the vagus; IO, inferior olive; NA, nucleus ambiguus; P, pyramidal tract; PH, nucleus praepositus hypoglossi; RB, restiform body; RFN, retrofacial nucleus; s, solitary tract; VN, vestibular nucleus; 5ST, spinal trigeminal tract; 5SP, spinal trigeminal nucleus; 7N, facial nucleus; 12N, hypoglossal nucleus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

source of inhibition of the medullary respiratory neurons and laryngeal motoneurons (Ezure and Manabe, 1988; Jiang and Lipski, 1990; Ono et al., 2006). Their inactivation may disinhibit laryngeal motoneurons and then help them to be active during vocalization (Sakamoto et al., 1996; Shiba et al., 2007). Some vocal-inactive E-AUG and E-CON neurons resumed firing when the vocal activity was attenuated at the last part of the stimulus-induced expiration. These types of neurons have been first described in the present study. Although their functional role has not been clarified, the cells may play a role in the termination of vocalization. The augmenting discharge of the phrenic nerve originates from rVRG I-AUG premotor neurons, the activity of which is guided by excitation from I-CON neurons, inhibition from I-DEC neurons, and self-reexcitation (Ezure, 1990). In the present study, the activity of most I-AUG neurons were closely correlated with that of the phrenic nerve during call site stimulation: the augmenting discharge of the phrenic nerve and augmenting firing of I-AUG neurons changed to the bell-shaped or plateau-like pattern. In contrast, the firing patterns of the I-DEC and I-CON neurons remained unchanged, although the discharge pattern of the phrenic nerve was altered during call site stimulation. These results indicate that

the PAG-induced phrenic discharge originates, at least in part, from I-AUG neurons and that additional neuronal inputs other than those from I-CON and I-DEC neurons are necessary for the PAG-induced activity of I-AUG neurons. The present study first showed that most phase-spanning neurons were strongly activated during vocalization. On the other hand, Larson et al. (1994) reported that many phase-spanning neurons are activated just before vocalization and only weakly activated during vocalization. This discrepancy may be due to the differences in experimental settings, because they used awake monkeys and classified the respiratory neuron types according to laryngeal muscle activity. Phase-spanning neurons are considered to be a source of respiratory phase switch, but their functional roles in breathing are still unclear (Cohen, 1969; Von Euler, 1986; Schwarzacher et al., 1995). The observations that many phasespanning neurons discharged during the vocal phase and ceased firing when the vocal activity was attenuated suggest that they contribute to the preservation of vocal emission as well as the phase transition. However, the connectivity between the phase-spanning neurons and the other brain stem respiratory neurons, including laryngeal motoneurons, remains unclarified. Further studies will be necessary to explore this possibility.

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Fig. 9. Behavior of respiratory neurons during swallowing. (A, B) These E-AUG and E-CON neurons were silent during swallowing identified by the swallow-related RLN burst induced by SLN stimulation (stim). (C) This E-DEC neuron was activated during swallowing. (D) This I-AUG neuron seemed to fire in synchrony with “swallow-breath” (indicated by the arrow). (E) This I-CON neuron discharged during the swallow-related RLN burst; the activity did not coincide with “swallow-breath” indicated by the arrow. (F) This IE neuron discharged during swallowing. (G) This IE neuron was silent during the swallow-related RLN burst. (H) This EI neuron began to fire approximately 0.3 s after the onset of the RLN burst. The thick horizontal bar at the bottom of each panel represents the stimulus duration of the SLN. Dashed vertical lines indicate the onset of the swallow-related RLN burst. The data in B and E were obtained from the same E-CON and I-CON neurons as in Figs. 4C and 6B, respectively.

Many investigators have reported that PAG stimulation first induces inspiration, followed by vocal emission, and that the vocal motor pattern consisting of the preceding inspiration and subsequent vocalization repeats during stimulation in cats and monkeys (Kirzinger and Jürgens, 1991; Larson et al., 1994; Shiba et al., 1996). The vocal motor pattern is preserved even after anesthetic application, decerebration, and paralyzation. However, the present study showed that the stimulation to the call site directly evoked the vocal activity without preceding inspiration in paralyzed anesthetized guinea pigs. We have previously reported that nonparalyzed guinea pigs also often begin PAG-induced vocalization from the vocal phase without a preceding inspiration (Sugiyama et al., 2010). Lung inflation is usually necessary for vocalization,

because limited air in the lung cannot sufficiently increase airflow or subglottic air pressure for vocalization (Nakazawa et al., 1997). The expiratory reserve volume of guinea pigs may be sufficient for vocalization so that the PAG stimulation first induces vocal phase. The present study has experimental limitations. First, we could not ascertain whether the cells were propriobulbar or bulbospinal because we did not employ antidromic stimulation to the spinal cord. Second, the data include some cranial motoneurons innervating the musculatures of upper airway or alimentary tract (Holstege et al., 1983; Altschuler et al., 1991). Indeed, some E-DEC neurons in the rVRG that were activated during swallowing also exhibited enhanced activity during vocalization and coughing, and are likely

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Fig. 10. Locations of respiratory neurons recorded during swallowing. Red symbols indicate neurons that were activated during swallowing, whereas black symbols designate neurons that were silent. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

to have profiles similar to pharyngeal motoneurons (Kirzinger and Jürgens, 1994; Ertekin and Aydogdu, 2003). 4.2. Swallowing Most E-AUG and E-CON neurons were silent during the swallowrelated RLN burst, whereas many E-DEC neurons were activated. These results are consistent with previous reports (Sumi, 1963; Oku et al., 1994; Saito et al., 2003). As discussed above, some E-DEC neurons recorded in the present study were probably motoneurons innervating the muscles of the upper aerodigestive tract, which facilitate a bolus transit during the pharyngeal swallowing. Inactivation of the E-AUG neurons whose firing inhibits these motoneurons can help to activate these motoneurons during swallowing (Ezure, 1990; Jiang and Lipski, 1990; Ono et al., 2006; Shiba et al., 2007). Most inspiratory neurons other than the onethird of I-AUG neurons were also silent during swallowing. Active inspiratory efforts during swallowing are fatal because of aspiration. Thus the cessation of the respiratory circuits responsible for inspiratory activity fits the physiological functions of swallowing with the exception of tongue muscle motoneurons and their premotor neurons with inspiratory-modulated activity (Ono et al., 1998; Gestreau et al., 2005). The other inspiratory upper airway motoneurons, i.e. pharyngeal and laryngeal motoneurons, are inactive during swallowing (Grélot et al., 1989; Shiba et al., 1999;

Gestreau et al., 2000). However, a minor inspiratory effort was occasionally observed during the pharyngeal stage of swallowing, referred to as “swallow-breath” (Fig. 9D and E) (Dick et al., 1993). The physiological role of swallow-breath is still unclear, but it is thought to assist the passage of the bolus from the pharynx to esophagus by generating negative intrathoracic pressure produced by minor inspiratory activity against a closed glottis (Shiba et al., 2007). Oku et al. (1994) also demonstrated that some rVRG I-AUG neurons fire during the period of “swallow-breath”. The present study also showed that the swallow-related firing of some I-AUG neurons occurred during “swallow-breath”. The evidence that most bulbospinal inspiratory neurons in the dorsal respiratory group are excited in synchrony with the “swallow-breath” (Gestreau et al., 1996; Saito et al., 2002) suggests that “swallow-breath” results from the dorsal respiratory group as well as from the ventrolateral medullary respiratory neurons. IE and EI neurons are suspected to function to initiate inspiratory and expiratory off-switches, respectively, by inhibiting various respiratory neurons (Bianchi and Gestreau, 2009). Here, approximately one-half of the IE neurons fired during swallowing. All recorded EI neurons were activated during swallowing, as shown by Zheng et al. (1997). These results raise the possibility that the activation of these phase-spanning neurons plays a role in the transient cessation of breathing during swallowing by suppressing the function of the medullary respiratory system.

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Fig. 11. Coughing-related activity of expiratory neurons. (A) An E-AUG neuron fired just after the bursting activity of the RLN during the expiratory phase of coughing presumably corresponding to the expulsive phase of coughing. (B) An E-AUG neuron was silent during fictive coughing. (C) An E-DEC neuron in the BötC was activated with a decrementing discharge pattern during the expiratory phase of coughing. Thick line at the bottom of each panel represents the stimulus duration of the RLN or tracheal mucosa (RLN stim or Trachea stim). Dashed lines indicate the respiratory phase transitions of coughing according to the PHR and ABD nerve activity.

4.3. Coughing The recorded E-AUG neurons exhibited various (active or inactive) responses during coughing throughout the expiratory phase of coughing. Because E-AUG neurons inhibit laryngeal adductor and phrenic motoneurons (Merrill and Fedorko, 1984; Jiang and Lipski, 1990; Ono et al., 2006), activation and inactivation of E-AUG neurons help to rest the diaphragm and close the glottis, respectively, during coughing (Shiba et al., 2007). The expiratory phase of coughing can usually be divided into the expulsive and narrowing phases: the glottis narrows against forced expiration to increase tracheal pressure during the compressive phase and then dilates to release a burst of air during the expulsive phase (Korpáˇs and Tomori, 1979). However, the coughing event itself or laryngeal motoneurons sometimes lack the expulsive phase (Shiba et al., 1999; Gestreau et al., 2000). Because the RLN includes both efferent axons of laryngeal closer and dilator motoneurons, it was sometimes impossible to determine whether or not the motor outputs corresponding to the expulsive phase were generated. When the cough-related RLN activity seemed to consist of the compressive and expulsive phases in some animals, as shown in Fig. 11A, EAUG neurons sharply fired during the presumptive expulsive phase. We think that this activation also facilitates the glottal opening by inhibiting laryngeal closer motoneurons during the expulsive phase (Shiba et al., 1999, 2007; Baekey et al., 2001). Although these cells were categorized as “activated”, some of these cells may be equivalent to the cells that were categorized as “silent” in animals whose cough-related RLN burst persisted throughout the abdominal burst. Most E-DEC neurons discharged during the expiratory phase of coughing, and their peak activities were observed near the onset of the expiratory phase of coughing (Fig. 11C). These

findings of expiratory neurons are consistent with the previous reports (Oku et al., 1994; Bongianni et al., 1998; Shannon et al., 2000). The rVRG E-DEC neurons that fired during coughing may correspond to the cranial motoneurons that innervate expiratory muscles (Ezure, 1990; Zheng et al., 1991; Bianchi et al., 1995). On the other hand, the BötC E-DEC neurons that were activated during coughing may contribute, at least in part, to the termination of inspiratory phase of coughing (Hayashi et al., 1996). Most I-AUG neurons and all other types of inspiratory neurons were activated during the inspiratory phase of coughing, and the firing patterns remained unchanged compared to those during normal respiration. As such, a majority of the inspiratory neurons probably act to shape the diaphragm activity during coughing, as do those during breathing. On the other hand, we found that a few I-AUG neurons fired during the expiratory phase of coughing. Some respiratory neurons classified as I-AUG neurons could include socalled “late-inspiratory neurons”, which fire in the late-inspiratory phase and facilitate the inspiratory off-switch by inhibiting other I-AUG neurons (Bianchi and Gestreau, 2009). In addition, some pharyngeal motoneurons exhibit late inspiratory activity (Grélot et al., 1989). All I-AUG neurons that were activated during the expiratory phase of coughing fired only at the end of the inspiratory phase of normal respiration. We consider these I-AUG neurons to be characterized as late-inspiratory neurons that inhibited other I-AUG neurons, suppressing an inspiratory effort during the expiratory phase of coughing, or as pharyngeal motoneurons. The phase-spanning neurons exhibited a variety of discharge patterns during coughing as reported by Shannon et al. (1998). All IE and half of the EI neurons fired during the expiratory phase of coughing; their peak firing rates occurred near the onset of the expiratory phase of coughing. In contrast, the other half of the EI

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Fig. 12. Firing of inspiratory neurons during coughing. (A–C) These I-AUG (A), I-DEC (B), and I-CON neurons (C) discharged during the inspiratory phase of coughing, whose firing patterns remained unchanged in comparison with those during normal respiration. (D) This late onset I-AUG neuron was activated during the expiratory phase of coughing. The data in A, B, and C were obtained from the same neuron as in Figs. 5A, 6A, and B, respectively.

neurons were silent during the expiratory phase of coughing. As discussed in other sections, it is possible that the excitation of the IE neurons at the beginning of the expiratory phase of coughing help the inspiratory off-switch and prevent inspiratory effort, and the EI neurons, possibly including the pharyngeal motoneurons,

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maintain upper airway patency during the expiratory phase of coughing. Meanwhile, the respiratory neurons located in the dorsolateral pons which are thought to be essential for producing respiratory rhythmogenesis (Alheid et al., 2004), involve many phase-spanning neurons whose discharge pattern is also

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Fig. 13. Activity of the phase-spanning neurons during coughing. (A) This IE neuron fired at the onset of the expiratory phase of coughing. (B) This EI neuron strongly discharged during the expiratory phase of coughing. Fluctuation of amplitude of spikes was attributed to the brainstem pulsation. (C) This EI neuron exhibited synchronous activity with the phrenic nerve burst during coughing, and became silent during the expiratory phase of coughing. The data in C were obtained from the same neuron as in Fig. 7B.

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Fig. 14. Locations of respiratory neurons with cough-related activity. For expiratory and phase-spanning neurons, red symbols indicate neurons that were activated during the expiratory phase of coughing, whereas black symbols designate neurons that were silent. For inspiratory neurons, red symbols indicate neurons that were activated during the inspiratory phase of coughing, while black symbols designate neurons that discharged during the expiratory phase of coughing. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

altered during coughing (Shannon et al., 2004). Furthermore, crosscorrelation analysis has revealed that phase-spanning neurons in the medulla are correlated with those in the pons (Segers et al., 1985). As such, it is also feasible that some interaction between phase-spanning neurons in the rostral ventrolateral medulla and those in the pons may exist and thereby facilitate the phase transition of coughing. However, further studies are necessary to assess the physiological role of the phase-spanning neurons during coughing. 4.4. Summary of activity of each type of respiratory neurons during vocalization, swallowing, and coughing The present study showed that the specific response patterns of each type of respiratory neurons were observed during vocalization, swallowing, and coughing. The E-AUG neurons that inhibit the upper airway motoneurons were generally silent during vocalization, swallowing, and the compressive phase of coughing. This inactivation of these cells may facilitate the activity of their motoneurons. Many E-DEC neurons possibly including the cranial motoneurons were activated during all behaviors tested. Many ECON neurons were activated during vocalization and coughing, but did not discharge during swallowing. The I-AUG neurons were typically activated in synchrony with the phrenic discharge during vocalization and coughing. On the contrary, some “late-inspiratory

neurons” discharged during the expiratory phase of coughing, probably contributing to the inspiratory–expiratory phase transition during coughing. The I-AUG neurons corresponding to phrenic premotor neurons fired during swallowing, suggesting that these neurons participate in the generation of “swallow-breath”. The discharge patterns of I-DEC neurons remained unchanged during the inspiratory phase of vocalization and coughing, while these neurons were silent during swallowing. The I-CON neurons were activated during vocalization and coughing. One swallow-active I-CON neuron was not in synchrony with “swallow-breath”. The physiological role of this type of neurons needs to be explored. Many phase-spanning neurons were activated during vocalization, swallowing, and coughing, which may play a role in the phase transition. In addition, the strong activation of these neurons during the vocal phase may play a key role in the preservation of the vocal activity, whereas the activation during swallowing may inhibit respiration. On the other hand, the EI neurons, some of which could be the pharyngeal motoneurons, may help to keep the pressure of forceful coughing. 4.5. Conclusions and perspectives We showed that respiratory neurons in the rostral ventrolateral medulla changed their activities in synchrony with non-respiratory behaviors including vocalization, swallowing, and coughing. As

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proposed by several researchers (Marder and Thirumalai, 2002; Nusbaum and Beenhakker, 2002), the neural circuitry that governs breathing can be altered by the afferent inputs that drive other motor patterns, resulting in the successful assembly of a specific pattern of respiratory movement. This flexible reconfiguration of the respiratory neuronal networks is possibly due to the modulated activity of individual respiratory neurons by altering their physiological property. This study suggests that signals that evoke the non-respiratory behaviors such as vocalization, swallowing, and coughing act as neuromodulators and alter the activity of respiratory neurons in a specific manner which is adjustable to provide each behavior. However, the mechanism underlying these behaviors cannot be fully explained only by this flexible activity of respiratory neurons. Although the respiratory neuronal networks may be involved in the central pattern generators of these nonrespiratory behaviors, these pattern generators primarily consist of their own individual neuronal networks. Our data thus support the view that the medullary respiratory neurons are multifunctional and can be shared in the motor neuronal circuits underlying these non-respiratory behaviors. Our findings have significant implications for understanding the mechanisms of the respiratory central pattern generator which regulates these non-respiratory behaviors. Grants This work was supported by Grant-in-Aid for Young Scientists (B) Grant Number 25861581 and Scientific Research (B) Grant Number 25293350. Acknowledgements The authors thank Dr. Ken Nakazawa, and Dr. Takeshi Suzuki for comments on an earlier version of the manuscript and Dr. Takeshi Nishio for assistance in data collection. References Alheid, G.F., Milsom, W.K., McCrimmon, D.R., 2004. Pontine influences on breathing: an overview. Respir. Physiol. Neurobiol. 143, 105–114. Altschuler, S.M., Bao, X.M., Miselis, R.R., 1991. Dendritic architecture of nucleus ambiguus motoneurons projecting to the upper alimentary tract in the rat. J. Comp. Neurol. 309, 402–414. Baekey, D.M., Morris, K.F., Gestreau, C., Li, Z., Lindsey, B.G., Shannon, R., 2001. Medullary respiratory neurones and control of laryngeal motoneurones during fictive eupnoea and cough in the cat. J. Physiol. 534, 565–581. Bianchi, A.L., Gestreau, C., 2009. The brainstem respiratory network: an overview of a half century of research. Respir. Physiol. Neurobiol. 168, 4–12. Bianchi, A.L., Denavitsaubie, M., Champagnat, J., 1995. Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters. Physiol. Rev. 75, 1–45. Bolser, D., 1991. Fictive cough in the cat. J. Appl. Physiol. 71, 2325–2331. Bongianni, F., Mutolo, D., Fontana, G.A., Pantaleo, T., 1998. Discharge patterns of Bötzinger complex neurons during cough in the cat. Am. J. Physiol. Regul. Integr. Comp. Physiol. 274, R1015–R1024. Cohen, M.I., 1969. Discharge patterns of brain-stem respiratory neurons during Hering–Breuer reflex evoked by lung inflation. J. Neurophysiol. 32, 356–374. De Lanerolle, N.C., 1990. A pontine call site in the domestic cat: behavior and neural pathways. Neuroscience 37, 201–214. Dick, T.E., Oku, Y., Romaniuk, J.R., Cherniack, N.S., 1993. Interaction between central pattern generators for breathing and swallowing in the cat. J. Physiol. 465, 715–730. Ertekin, C., Aydogdu, I., 2003. Neurophysiology of swallowing. Clin. Neurophysiol. 114, 2226–2244. Ezure, K., 1990. Synaptic connections between medullary respiratory neurons and considerations on the genesis of respiratory rhythm. Prog. Neurobiol. 35, 429–450. Ezure, K., Manabe, M., 1988. Decrementing expiratory neurons of the Bötzinger complex. II. Direct inhibitory synaptic linkage with ventral respiratory group neurons. Exp. Brain Res. 72, 159–166. Feldman, J.L., Del Negro, C.A., 2006. Looking for inspiration: new perspectives on respiratory rhythm. Nat. Rev. Neurosci. 7, 232–242.

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