Respiratory Physiology & Neurobiology 203 (2014) 90–97

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Vagal afferent control of abdominal expiratory activity in response to hypoxia and hypercapnia in rats Eduardo V. Lemes a , Daniel B. Zoccal a,b,∗ a b

Department of Physiological Sciences, Center of Biological Sciences, Federal University of Santa Catarina (UFSC), Florianópolis, SC, Brazil Department of Physiology and Pathology, School of Dentistry of Araraquara, São Paulo State University (UNESP), Araraquara, SP, Brazil

a r t i c l e

i n f o

Article history: Accepted 16 August 2014 Available online 10 September 2014 Keywords: Active expiration Electromyography Hypercapnia Hypoxia Vagotomy Pulmonary stretch receptors

a b s t r a c t In the present study, we tested the hypothesis that vagal afferent information modulates the pattern of expiratory response to hypercapnia and hypoxia. Simultaneous recordings of airflow, diaphragmatic (DIA) and oblique abdominal muscle (ABD) activities were performed in anesthetized (urethane, 1.2 g/kg), tracheostomized, spontaneously breathing male Wistar rats (290–320 g, n = 12). The animals were exposed to hypercapnia (7 and 10% CO2 for 5 min) and hypoxia (7% O2 for 1 min) before and after bilateral vagotomy. We verified that the percentage increase in DIA burst amplitude elicited by hypercapnia and hypoxia episodes was similar between intact and vagotomized rats (P > 0.05). In contrast, hypercapnia and hypoxia promoted a marked increase in ABD activity in vagotomized, but not in intact rats (P < 0.01). These amplified expiratory motor changes after vagotomy were associated with enhanced expiratory airflow (P < 0.01) and augmented tidal volume responses (P < 0.01). Our data indicates that, in anesthetized conditions, the removal of peripheral afferent inputs facilitates the processing of active expiration in response to hypercapnia and hypoxia in rats. © 2014 Elsevier B.V. All rights reserved.

1. Introduction In mammals, breathing results from the synchronized activity of cranial and spinal motor nerves that drive periodic contractions of respiratory muscles (Richter and Smith, 2014). In conditions of normoxia and normocapnia, inspiration begins with the contraction of inspiratory muscles whilst expiration occurs passively due to the recoil forces of the lungs and the chest (St-John and Paton, 2003). The inspiratory/expiratory phase switching is determined by the coordinated activity of neurons of the respiratory central pattern generator (CPG) located in the lower brainstem (Richter and Smith, 2014; Rybak et al., 2004a; Smith et al., 2007). In the ventrolateral surface of medulla oblongata, the inspiratory neurons of the pre-Bötzinger complex (pre-BötC) are considered essential for the inspiratory rhythm generation (Smith et al., 1991; Tan et al., 2008). The pre-BötC inspiratory neurons are suggested to interact with the expiratory neurons of the Bötzinger complex (BötC) and form the core of the respiratory CPG (Rybak et al., 2004b; Smith et al.,

∗ Corresponding author at: Department of Physiology and Pathology, School of Dentistry of Araraquara, São Paulo State University (UNESP), Rua Humaitá, 1680, Araraquara, SP, CEP: 14801-903, Brazil. Tel.: +55 16 3301 6555; fax: +55 16 33016488. E-mail addresses: [email protected], [email protected] (D.B. Zoccal). http://dx.doi.org/10.1016/j.resp.2014.08.011 1569-9048/© 2014 Elsevier B.V. All rights reserved.

2007). This core establishes reciprocal synaptic connections with other pontine and medullary respiratory compartments, including the dorsolateral pons (Dobbins and Feldman, 1994; Molkov et al., 2013), dorsal respiratory group (Alheid et al., 2011; de Castro et al., 1994) and chemosensitive nuclei (Biancardi et al., 2008; Rosin et al., 2006), whose integrity is essential for the generation of the eupneic breathing pattern (Costa-Silva et al., 2010; Richter, 1982; Richter and Spyer, 2001; Smith et al., 2007). Sensory afferents located in the lungs, airways and carotid bodies provide a powerful feedback information to respiratory CPG and contribute to shape the breathing pattern (Hayashi et al., 1996; Janczewski et al., 2013; Kubin et al., 2006; Moraes et al., 2012a). Afferent inputs from pulmonary stretch receptors, mainly from the slowly adapting receptors (SARs), modulate central respiratory activity according to the lung volume (Kubin et al., 2006). The activation of SARs vagal afferents suppresses the inspiratory motor activity and prolongs the expiratory phase – representing the socalled Hering-Breuer reflex (Backman et al., 1984; Bonham et al., 1993). The removal of pulmonary afferents, by bilateral vagotomy, promotes a marked increase in baseline inspiratory amplitude as well as in the inspiratory and expiratory phase durations, supporting the concept that the activation of SARs vagal afferents importantly contributes to inspiratory/expiratory phase transition during eupneic breathing (Molkov et al., 2013; Mörschel and Dutschmann, 2009).

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Peripheral and central chemoreceptors adjust the breathing pattern according to the levels of oxygen (O2 ) and carbon dioxide (CO2 ) in the arterial blood, respectively (Costa-Silva et al., 2010; Powell et al., 1998; Takakura et al., 2014). In conditions of hypoxia and hypercapnia, increases in the inspiratory motor activity occur to enhance respiratory frequency and tidal volume (Powell et al., 1998). In addition, the stimulation of peripheral and central chemoreceptors also transforms expiration into an active process, promoting the emergence of periodic contractions in the abdominal expiratory muscles (Abdala et al., 2009a; Moraes et al., 2012a). Therefore, the ventilatory responses to hypoxia and hypercapnia are associated with increments in both inspiratory and expiratory motor activities. However, the experimental evidence supporting this notion was obtained in anesthetized-vagotomized ventilated rats (Marina et al., 2010) and in the decerebrated in situ preparations (Abdala et al., 2009a; Moraes et al., 2012a), in which pulmonary vagal afferents are absent. Previous studies performed in anesthetized cats reported that abdominal expiratory muscle contractions are engaged when lung deflation is hindered (Marek et al., 2008), suggesting that interactions between pulmonary afferent pathways and the mechanisms responsible for the generation of active expiration may occur. Based on that, in the present study we hypothesized that the pattern of ventilatory response to hypercapnia and hypoxia may modify after the removal of vagal feedback information. Specifically, we considered the possibility that the activation of vagal afferents during hypoxic and hypercapnic episodes modulates the amplitude of evoked expiratory motor activity. Therefore, herein we investigated the effects of bilateral vagotomy on the pattern of inspiratory and expiratory motor responses to hypercapnia and hypoxia. 2. Materials and methods 2.1. Animals and ethical approval Experiments were performed on male Wistar rats (n = 18), weighing 290–320 g, obtained from the Federal University of Santa Catarina Animal Breeding Center and kept at 22 ± 1 ◦ C on a 12-h light/dark cycle (lights on 06:00 – lights off 18:00), with access to food and water ad libitum. All experimental procedures followed the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23 revised 1996) and by the Brazilian National Council for Animal Experimentation Control (CONCEA), and were approved by the Local Ethical Committee in Animal Experimentation (protocol PP00543). 2.2. Experimental preparation Surgical procedures and experiments were performed under anesthesia with urethane (1.2 g/kg, i.p.). The level of anesthesia was constantly assessed by the absence of corneal and toe-pinch withdrawal reflexes. Additional doses of urethane were administrated (10–20% of initial dose), when necessary, to maintain anesthesia in adequate levels. Body temperature was maintained at 36–38 ◦ C. Animals were positioned supine and a cervical incision was made to expose the trachea and isolate right and left vagus nerves. The rats were then tracheostomised and the tracheal cannula was connected to a 3-way stopcock, which allowed the simultaneous monitoring of airflow and the administration of gas mixtures. Animals breathed spontaneously and were maintained at 100% oxygen (O2 ) during surgery and experimental protocols, except when exposed to hypercapnic or hypoxic episodes. Polyethylene catheters (PE-50 connected to PE-10; Clay Adams, Parsippany, NJ, USA) were inserted into the right femoral artery and vein for

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arterial pressure measurements and systemic administration of fluids, respectively. Bipolar stainless steel electrodes were implanted in the diaphragm (DIA) and in the oblique abdominal muscles (ABD) to perform electromyographic (EMG) recordings of inspiratory and expiratory motor activities, respectively. To minimize deviations in blood pH and maintain fluid balance, slow intravenous administration of Ringer’s solution (in mM: 125 NaCl, 24 NaHCO3 , 3 KCl, 2.5 CaCl2 , 1.25 MgSO4 , 1.25 KH2 PO4 , 10 dextrose) containing lactate (2 mM) was performed (3–4 ml/kg/h) during the experiments (MacFarlane and Mitchell, 2009; Xing and Pilowsky, 2010). Before starting the experimental protocols, a period of at least 30 min was allowed for stabilization. 2.3. Recordings of cardiorespiratory parameters Inspiratory and expiratory airflows were evaluated using a differential pressure transducer (ML141, ADInstruments, Bella Vista, NSW, Australia) connected to the tracheal cannula. Pulsatile arterial pressure (PAP) was measured by connecting the arterial catheter to a pressure transducer (MLT0380, ADInstruments) that, in turn, was connected to an amplifier (ML221 Bridge Amp, ADInstruments). Airflow and PAP signals were acquired by a data acquisition system (Powerlab, ADInstruments) and recorded on a computer (sampling rate of 1 kHz per channel) using appropriated software (LabChart, ADInstruments). Values of mean arterial pressure (MAP, mmHg) and heart rate (HR, bpm) were derived from PAP signals. DIA and ABD signals were amplified (Bioamplifier, Insight, Ribeirão Preto, SP, Brazil), band-pass filtered (0.1–2 kHz) and acquired at a sampling rate of 2 kHz per channel (LabChart, ADInstruments). Respiratory motor outputs and cardiovascular parameters were recorded simultaneously. 2.4. Experimental protocol Baseline cardiorespiratory parameters were recorded initially for 15–20 min. The animals were then exposed to hypercapnic (7% and 10% of inspired carbon dioxide, CO2 , balanced in O2 , for 5 min) and hypoxic episodes (7% of inspired O2 , balanced in nitrogen, N2 , for 1 min) and the cardiorespiratory reflex responses were recorded. The hypercapnic and hypoxic gases were administrated to the animals through the tracheal cannula, using a gas mixture device (AVS Projetos, São Carlos, Brazil) coupled to cylinders of 100% of O2 , CO2 and N2 (White Martins, Florianópolis, Brazil) and to a gas analyzer (ML206 Gas Analyzer, ADInstruments). The hypercapnic and hypoxic stimuli were applied randomly and a minimum period of 10 min was given between consecutive stimuli. After these procedures, left and right vagus nerves were cut at the cervical level (below the carotid bifurcation) and a 15–20 min period was allowed for stabilization. Baseline and evoked cardiorespiratory parameters were then recorded as aforementioned. At the end of the experiments, animals were euthanized with intravenous injections of KCl 10%. 2.5. Data analysis The EMG signals were rectified and smoothed (50 ms) for analysis. DIA motor activity was evaluated by its burst amplitude (mV), frequency (referred as respiratory frequency, fR, and expressed in cycles per minute, cpm) and duration (time of inspiration, ms). The time between consecutive bursts was also determined (time of expiration, ms). ABD motor activity was measured by its burst amplitude (mV). Tidal volume (VT ) was calculated from the inspiratory and expiratory flows (ml/s) and normalized by the animal weight (ml/kg). The effects of bilateral vagotomy on baseline inspiratory and expiratory flows, MAP, HR, DIA and ABD activities were assessed by the comparison of average values measured before

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and after vagotomy during baseline period (prior to the stimuli). The magnitudes of the cardiorespiratory changes elicited by hypercapnia and hypoxia were calculated as the difference between the evoked response (measured as the average of the final 15–30 s of the first half of the exposure period) and baseline values (measured immediately before the respective stimulus). Evoked DIA and ABD burst amplitudes were expressed as percentage values (%) in relation to basal activity while the other parameters were expressed in their original units. All analyses were performed offline using LabChart software. 2.6. Statistics The data were expressed as mean ± standard error of mean (SEM). Before analyses, data distribution was checked using the Shapiro–Wilk normality test. The effects of bilateral vagotomy on the cardiorespiratory responses evoked by hypercapnia and hypoxia were then compared using paired Student’s t test, with exception of the DIA and ABD burst amplitude data that were compared using Wilcoxon matched pairs test (nonparametric data). The comparisons were carried out using GraphPad Prism software (version 5, La Jolla, CA, USA) and differences were considered significant at P < 0.05. 3. Results 3.1. Baseline cardiovascular and respiratory changes elicited by bilateral vagotomy The effects of bilateral vagotomy on respiratory motor outputs and cardiovascular parameters are shown in Fig. 1. After vagotomy, the rats (n = 18) exhibited: (i) augmented DIA burst amplitude (20.6 ± 2.3 vs 31.9 ± 4.2 mV, respectively intact and vagotomized rats, P < 0.001, Fig. 1C); (ii) reduced respiratory frequency (96 ± 4 vs 44 ± 2 cpm, respectively intact and vagotomized rats, P < 0.001, Fig. 1D); (iii) enhanced inspiratory flow (−4.8 ± 0.3 vs −5.9 ± 0.3 ml/s, respectively intact and vagotomized rats, P < 0.001), expiratory flow (5.7 ± 0.2 vs 7.9 ± 0.3 ml/s, respectively intact and vagotomized rats, P < 0.001) and tidal volume (2.4 ± 0.3 vs 3.8 ± 0.4 ml/kg, respectively intact and vagotomized rats, P < 0.001, Fig. 1E); and (iv) prolonged times of inspiration (461 ± 23 vs 884 ± 36 ms, respectively intact and vagotomized rats, P < 0.001) and expiration (190 ± 22 vs 521 ± 46 ms, respectively intact and vagotomized rats, P < 0.001, Fig. 1F). Bilateral vagotomy did not promote significant changes in baseline ABD motor activity (2.6 ± 0.5 vs 2.3 ± 0.3 mV, respectively intact and vagotomized rats, Fig. 1C), MAP (107 ± 5 vs 104 ± 4 mmHg, respectively intact and vagotomized rats) and HR (380 ± 9 vs 372 ± 12 bpm, respectively intact and vagotomized rats). 3.2. Cardiorespiratory responses to hypercapnia and hypoxia before and after bilateral vagotomy Fig. 2 illustrates the pattern of respiratory responses induced by hypercapnia before and after bilateral vagotomy. The group data are summarized in Table 1. Before vagotomy, hypercapnia (7 and 10% CO2 , n = 12) increased DIA burst amplitude (P < 0.001, Fig. 4A), inspiratory and expiratory flows (P < 0.001, Fig. 4B and C) and tidal volume (P < 0.001, Fig. 4D). Respiratory frequency (Fig. 4E) and ABD burst amplitude (Fig. 4F) did not present any significant changes. With respect to cardiovascular parameters, hypercapnia did not produce significant alterations in MAP, but promoted a reduction in HR (P < 0.01). After bilateral vagotomy, the pattern of ventilatory responses to hypercapnia modified significantly. Although the hypercapnic-induced increase in DIA burst amplitude exhibited higher absolute values (mV) in vagotomized than in intact

Table 1 Average changes in cardiorespiratory parameters induced by hypercapnia (7 and 10% CO2 for 5 min) and hypoxia (7% O2 for 1 min) in anesthetized rats before (intact rats) and after bilateral vagotomy (vagotomized rats). ns – not significant (P > 0.05); *, ** and ***Different from intact group, respectively P < 0.05, P < 0.01, P < 0.001. Abbreviations – DIA: diaphragm; fR: respiratory frequency; VT : tidal volume; ABD: abdominal; MAP: mean arterial pressure; HR: heart rate. Intact rats

Vagotomized rats

7% CO2 (n = 12) DIA burst amplitude (%) fR (cpm) Inspiratory flow (ml/s) Expiratory flow (ml/s) VT (ml/kg) ABD burst amplitude (%) MAP (mmHg) HR (bpm)

36 0 −13.8 10.8 5.1 2 3 −18

± ± ± ± ± ± ± ±

4 2 3.4 0.8 0.9 1 4 3

58 3 −26.6 20.8 14.3 21 5 −16

± ± ± ± ± ± ± ±

15ns 4ns 4.7*** 2*** 3.4*** 5** 3ns 5ns

10% CO2 (n = 12) DIA burst amplitude (%) fR (cpm) Inspiratory flow (ml/s) Expiratory flow (ml/s) VT (ml/kg) ABD burst amplitude (%) MAP (mmHg) HR (bpm)

69 −4 −18.6 11.1 8.2 4 −2 −31

± ± ± ± ± ± ± ±

12 4 3.9 1.7 1.6 2 3 3

95 −6 −28.6 20.5 15.9 47 0 −24

± ± ± ± ± ± ± ±

20ns 1ns 4.6** 2.1*** 3.4** 8*** 4ns 10ns

7% O2 (n = 6) DIA burst amplitude (%) fR (cpm) Inspiratory flow (ml/s) Expiratory flow (ml/s) VT (ml/kg) ABD burst amplitude (%) MAP (mmHg) HR (bpm)

31 39 −7.38 7.5 3.4 19 −65 −16

± ± ± ± ± ± ± ±

10 6 0.9 0.5 0.4 6 7 9

51 14 −12.8 15.6 8.1 63 −55 −11

± ± ± ± ± ± ± ±

8ns 2** 1.1* 1.3*** 1.2** 15* 7ns 9ns

rats (Fig. 2C and D), the percentage increase (relative to respective baseline) was not different between groups (Fig. 4A). On the other hand, vagotomized rats displayed a notable increase in ABD expiratory motor activity during hypercapnia, which was not seen in intact animals (Fig. 2C and D; Fig. 4F; P < 0.01). Bilateral vagotomy also amplified the responses of increase in inspiratory flow, expiratory flow and tidal volume (Fig. 4B–D; P < 0.05). No changes were observed in the respiratory frequency (Fig. 4E) and in the cardiovascular responses to hypercapnia after vagotomy. The pattern of ventilatory responses to hypoxia also changed after vagotomy, as demonstrated in Fig. 3. In intact animals, the exposure to 7% of O2 increased: (i) DIA burst amplitude (Fig. 4A, P < 0.01); (ii) inspiratory and expiratory flows (Fig. 4B and C, P < 0.01); (iii) tidal volume (Fig. 4D, P < 0.001); (iv) respiratory frequency (Fig. 4E, P < 0.01); and (v) ABD burst amplitude (Fig. 4F; P < 0.05). Hypoxia exposure also promoted a large fall in MAP (P < 0.01) and no significant changes in HR. After bilateral vagotomy, the percentage increase in DIA burst amplitude in response to hypoxia was similar to that observed in intact conditions (Figs. 3C, D and 4A). On the other hand, the responses of increase in inspiratory flow (Fig. 4B, P < 0.05), expiratory flow (Fig. 4C, P < 0.001), tidal volume (Fig. 4D, P < 0.01) and ABD burst amplitude (Fig. 4F, P < 0.05) were amplified in vagotomized rats (Fig. 4F, P < 0.05). In contrast, the evoked increase in respiratory frequency was smaller after vagotomy (P < 0.01, Fig. 4E). The magnitude of cardiovascular responses to hypoxia was similar between vagotomized and intact rats. The group data are summarized in Table 1. 4. Discussion It is well known that afferent inputs from pulmonary stretch receptors are an important feedback mechanism that modulates central inspiratory activity and inspiratory/expiratory phase

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A HR (bpm)

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B

390 355 320 150

PAP 100 (mmHg)

5 mV

50

∫ABDEMG ABDEMG

5 mV

∫DIAEMG DIAEMG Flow (mL/s)

5 0 -5

1s

C

D

1s

E

F

Fig. 1. Effects of bilateral vagotomy on baseline cardiorespiratory parameters of anesthetized  animals. Panels A and B: Recordings of the heart rate (HR), pulsatile arterial pressure (PAP), abdominal (ABD) and diaphragmatic (DIA) activities [raw and integrated ( )] and airflow from a representative rat of the group, illustrating baseline cardiorespiratory parameters before (A) and after (B) bilateral vagotomy. Panels C–F: Average changes induced by bilateral vagotomy in baseline DIA and ABD burst amplitude (C), respiratory frequency (fR, D), tidal volume (VT , E) and times of inspiration and expiration (F) of anesthetized rats (n = 18). ***Different from intact animals (P < 0.001).

transition (Kubin et al., 2006). Our observations that bilateral vagotomy increased diaphragm burst amplitude and prolonged the times of inspiration and expiration are in agreement with the notion that pulmonary vagal afferent information contribute significantly for the generation of eupneic breathing pattern. In addition, in the present study we verified that vagal afferent inputs also play a relevant role in the patterning of respiratory responses to hypercapnia and hypoxia. Our most evident observation was that vagotomized rats exposed to hypercapnia (7% and 10% CO2 ) and hypoxia (7% O2 ) exhibited reflex responses of contractions in abdominal muscles – a response that was not seen in animals with intact vagus nerves. These findings suggest that, in conditions of anesthesia, the pulmonary afferent inputs negatively modulate the mechanisms responsible for the generation of active expiration. Lung inflation initiates the Hering-Breuer reflex and promotes the inhibition of inspiratory activity and the lengthening of expiratory phase (Kubin et al., 2006). These reflex respiratory responses are triggered by the activation of slowly adapting pulmonary stretch receptors (SARs), whose axons project to the brainstem through the vagus nerves and make synapses within the nucleus of the solitary tract (NTS) with a group of 2nd-order neurons called pump-cells (Bonham et al., 1993). The pump-cells are suggested to integrate and transmit the pulmonary afferent information to respiratory neurons of the pons and ventral medulla (Ezure et al., 2002; Kanbar et al., 2011; McCrimmon et al., 1987). Studies have proposed that the respiratory responses of Hering-Breuer reflex are mediated, at least in part, by the activation of BötC post-inspiratory (E-decrementing) neurons (Ezure et al., 2002; Hayashi et al., 1996) that, in turn, suppress the activity of medullary inspiratory neurons (Janczewski et al., 2013; Molkov et al., 2013; Tian et al., 1999).

Based on that, the increased inspiratory motor activity and the augmented tidal volume observed after bilateral vagotomy may be due to a depression of post-I activity and consequent disinhibition of inspiratory neurons (Molkov et al., 2013). Although baseline inspiratory activity was enhanced after vagotomy, the removal of vagal afferents may have not altered significantly the hypercapnic- and hypoxic-induced excitatory drives to inspiratory neurons because the percentage increase in DIA burst amplitude (in relation to baseline amplitude) was similar between vagotomized and intact rats. In contrast, the evoked expiratory motor response to hypercapnia and hypoxia was markedly amplified after bilateral vagotomy, since vagotomized, but not intact rats, exhibited a clear abdominal expiratory contractile response when submitted to these stimuli. Importantly, bilateral vagotomy did not induce any changes in baseline abdominal motor activity, suggesting that the removal of vagal afferent information facilitates the processing of expiratory motor activity in response to hypercapnia and hypoxia. Accumulating evidence suggests that the parafacial respiratory group (pFRG) – a region located in the ventral to the facial nucleus and rostral to the BötC, overlapped with the retrotrapezoid nucleus (RTN) – critically contributes for the generation of active expiration (Abdala et al., 2009a; Janczewski and Feldman, 2006; Moraes et al., 2012a). This region contains expiratory neurons that are silent at resting conditions, but exhibit rhythmic discharges during hypercapnia (Abdala et al., 2009a) or after the stimulation of peripheral chemoreceptors (Moraes et al., 2012a). It has been suggested that the activation of the RTN/pFRG expiratory neurons, and the emergence of expiratory motor activity, depends on a balance between excitatory and inhibitory mechanisms (Marina et al., 2010; Molkov

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Fig. 2. Cardiorespiratory responses to hypercapnia before and after bilateral vagotomy. Recordings from an anesthetized rat, representative of the group, showing the pattern of cardiovascular and respiratory changes induced by hypercapnia (10% CO2 for 5 min, beginning indicated by the arrow) before (panel A) and after (panel B) bilateral vagotomy. Panel C and D: recordings extracted from panels A and B, respectively, demonstrating the DIA and ABD evoked responses to hypercapnia in expanded time scale. Note the emergence of the ABD response to hypercapnia after bilateral vagotomy.

Fig. 3. Cardiorespiratory responses to hypercapnia before and after bilateral vagotomy. Recordings from an anesthetized rat, representative of the group, showing the pattern of cardiovascular and respiratory changes induced by hypoxia (7% O2 for 1 min, beginning indicated by the arrow) before (panel A) and after (panel B) bilateral vagotomy. Panel C and D: recordings extracted from panels A and B, respectively, demonstrating the DIA and ABD evoked responses to hypoxia in expanded time scale. Note that after vagotomy, contractions of ABD occurred in response to hypoxia.

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Fig. 4. Respiratory responses to hypercapnia and hypoxia before and after bilateral vagotomy. Average changes of diaphragm (DIA) burst amplitude (A), inspiratory (B) and expiratory (C) flows, tidal volume (VT , D), respiratory frequency (fR) (E) and abdominal (ABD) burst amplitude (F) induced by hypercapnia (7 and 10% CO2 , n = 12) and hypoxia (7% O2 , n = 6) in urethane-anesthetized animals before (intact) and after bilateral vagotomy (vagotomized). *, ** and ***Different from intact group, respectively P < 0.05, P < 0.01, P < 0.001.

et al., 2010, 2011). Studies by Pagliardini et al. (2011) demonstrated that the antagonism of GABAergic and glycinergic neurotransmission in the RTN/pFRG evokes abdominal contractions, suggesting that the activity of expiratory neurons of this region is tonically suppressed by inhibitory inputs. According to Molkov et al. (2010), the inhibitory drive to the RTN/pFRG may arise mainly from the respiratory neurons of the ventral respiratory column, including the post-inspiratory neurons of the BötC. Accordingly, we hypothesized that the reflex abdominal muscle contractions were not observed in intact animals exposed to hypercapnia and hypoxia (at least in anesthetized conditions and under certain levels of CO2 and O2 ) because the concurrent SAR activation during these stimuli (consequent to increased inspiratory drive) increased the post-inspiratory neuronal activity in the BötC (Hayashi et al., 1996) that, in turn, may have suppressed the excitation of RTN/pFRG expiratory neurons by the peripheral and central chemoreceptor inputs. With the removal of vagal afferents, SAR-dependent activation of post-inspiratory neurons of the BötC during the hypercapnic and hypoxic stimuli was reduced, or even blunted. As a result, expiratory neurons of the RTN/pFRG were released from inhibition and the active expiratory pattern emerged in response to hypercapnia and hypoxia. Alternatively, the negative modulation of pulmonary afferent inputs on the active expiratory pattern may have occurred through the activation of inhibitory pump-cells of the NTS that send projections to the RTN/pFRG neurons (Takakura et al., 2007). Although these hypotheses still require further experimental evidence to be proven, the possible inhibitory interaction between pulmonary afferent feedback and the neuronal activity of RTN/pFRG is supported by studies by Moreira et al. (2007) reporting that lung deflation increases the firing rate of CO2 sensitive neurons with expiratory pattern of discharge whilst lung inflation suppresses their activity. In addition to the medullary mechanisms, the respiratory groups of the pons (Kölliker-Fuse, Parabrachial nucleus, A5 area and others) are also critical for the generation of active expiration (Abdala et al., 2009a,b). Studies have proposed that a SAR-dependent feedback loop from NTS pumpcells modulates negatively the activity of the pontine respiratory neurons (Molkov et al., 2013). Based on that, we may also suggest

that the emergence of evoked expiratory motor activity following vagotomy is associated with an increased pontine excitatory drive to the ventromedullary expiratory neurons. These possibilities still await experimental verification. A limitation of our study was the lack of blood gas measurements in intact and vagotomized animals during the exposure to hypercapnia and hypoxia. Since the levels of partial pressure of CO2 and O2 in arterial blood may have varied between intact and vagotomized rats, we cannot exclude the possibility that part of the effects of vagotomy on evoked expiratory activity was related to the differences in the chemical excitatory drive to expiratory neurons. However, considering our observations that intact animals presented only minor increases in expiratory activity at the highest hypercapnic stimulus administrated (10% CO2 ) while vagotomized animals already exhibited a clear increase in abdominal burst amplitude at the smallest hypercapnic stimulus (7% CO2 ), we suggest that possible differences in chemoreceptor-induced excitatory drive did not contribute significantly to the facilitation in expiratory motor activity after vagotomy. In vagotomized animals, the maximal increase in minute ventilation in response to hypercapnia and hypoxia is expected to be smaller than in intact animals due to the large decrease in baseline respiratory frequency. With the recruitment of abdominal muscles, the expiratory reserve volume may have been accessed after vagotomy, then increasing the amplitude of evoked tidal volume responses in order to compensate the reduction in respiratory frequency. Therefore, the generation of active expiratory pattern in response hypoxia and hypercapnia may represent a critical mechanism to amplify the responses of increase in tidal volume, preventing any significant decrease in minute ventilation consequent to vagotomy. However, future studies are required to better elucidate the functional role of active expiration in rats. Different from the ventilatory responses, the cardiovascular reflex changes induced by hypercapnia and hypoxia were similar in intact and vagotomized conditions. Based on studies showing that elevated sympathetic activity and arterial pressure levels are observed coupled to the emergence of active expiration in experimental models of chronic hypoxia (Moraes et al., 2013, 2014;

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Zoccal et al., 2008), we hypothesized that vagotomy, and the consequent facilitation of evoked expiratory motor response, would modify the magnitude of cardiovascular responses to hypoxia and hypercapnia, due to central interactions between expiratory and sympathetic/parasympathetic neurons (Moraes et al., 2012b; Zoccal et al., 2009). However, the absence of changes in the average arterial pressure and heart rate responses do not rule out the possibility that the pattern of evoked sympathetic and parasympathetic discharges to cardiovascular system may have been modified with the generation of active expiration following vagotomy. Therefore, additional experiments involving direct nerve recordings will be required for a better evaluation of the effects of vagotomy on the processing of autonomic responses to hypoxia and hypercapnia. 5. Conclusion In the present study, we reported that anesthetized rats lacking the vagal afferent feedback (due to bilateral vagotomy) showed a different pattern of ventilatory response to hypercapnia and hypoxia in comparison to animals with intact vagus nerve. Specifically, vagotomized rats exposed to hypercapnia and hypoxia exhibited clear amplified contractions in abdominal expiratory muscles, which were not seen in intact animals. These findings indicate that vagal afferent inputs, including those from pulmonary stretch receptors, modulate negatively the emergence of active expiratory pattern in conditions of hypercapnia and hypoxia. Contributors DBZ designed the experimental protocols. EVL performed the experiments. DBZ and EVL analyzed the data, drafted the manuscript and approved its final version. Acknowledgments This study was supported by Fundac¸ão de Amparo à Pesquisa e Inovac¸ão Científica do Estado de Santa Catarina (FAPESC, grant #24.495/2010-3) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant # 151402/2012). The authors greatly thank Dr. Benedito H. Machado from his important and helpful support for the initial steps of this study. References Abdala, A.P., Rybak, I.A., Smith, J.C., Paton, J.F., 2009a. Abdominal expiratory activity in the rat brainstem-spinal cord in situ: patterns, origins and implications for respiratory rhythm generation. J. Physiol. 587, 3539–3559. Abdala, A.P., Rybak, I.A., Smith, J.C., Zoccal, D.B., Machado, B.H., St-John, W.M., Paton, J.F., 2009b. Multiple pontomedullary mechanisms of respiratory rhythmogenesis. Respir. Physiol. Neurobiol. 168, 19–25. Alheid, G.F., Jiao, W., McCrimmon, D.R., 2011. Caudal nuclei of the rat nucleus of the solitary tract differentially innervate respiratory compartments within the ventrolateral medulla. Neuroscience 190, 207–227. Backman, S.B., Anders, C., Ballantyne, D., Rohrig, N., Camerer, H., Mifflin, S., Jordan, D., Dickhaus, H., Spyer, K.M., Richter, D.W., 1984. Evidence for a monosynaptic connection between slowly adapting pulmonary stretch receptor afferents and inspiratory beta neurones. Pflugers Arch. 402, 129–136. Biancardi, V., Bícego, K., Almeida, M., Gargaglioni, L., 2008. Locus coeruleus noradrenergic neurons and CO2 drive to breathing. Pflugers Arch. Eur. J. Physiol. 455, 1119–1128. Bonham, A.C., Coles, S.K., McCrimmon, D.R., 1993. Pulmonary stretch receptor afferents activate excitatory amino acid receptors in the nucleus tractus solitarii in rats. J. Physiol. 464, 725–745. Costa-Silva, J.H., Zoccal, D.B., Machado, B.H., 2010. Glutamatergic antagonism in the NTS decreases post-inspiratory drive and changes phrenic and sympathetic coupling during chemoreflex activation. J. Neurophysiol. 103, 2095–2106. de Castro, D., Lipski, J., Kanjhan, R., 1994. Electrophysiological study of dorsal respiratory neurons in the medulla oblongata of the rat. Brain Res. 639, 49–56. Dobbins, E.G., Feldman, J.L., 1994. Brainstem network controlling descending drive to phrenic motoneurons in rat. J. Comp. Neurol. 347, 64–86. Ezure, K., Tanaka, I., Saito, Y., Otake, K., 2002. Axonal projections of pulmonary slowly adapting receptor relay neurons in the rat. J. Comp. Neurol. 446, 81–94.

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