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ARTICLE IN PRESSG ModelESPNB 2499 1–9

Respiratory Physiology & Neurobiology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Respiratory Physiology & Neurobiology

jou rn al h om epa ge: www.elsev ier .com/ locate / resphys io l

ifferences in respiratory changes and Fos expression in theentrolateral medulla of rats exposed to hypoxia, hypercapnia, andypercapnic hypoxia

un Wakaia, Daichi Takamurab, Ryosuke Morinagac, Nobuaki Nakamutac,oshio Yamamotoc,∗

Laboratory Animal Research Center, Fukushima Medical University School of Medicine, Fukushima, JapanLaboratory of Veterinary Biochemistry and Cell Biology, Faculty of Agriculture, Iwate University, Morioka, JapanLaboratory of Veterinary Anatomy and Cell Biology, Faculty of Agriculture, Iwate University, Morioka, Japan

r t i c l e i n f o

rticle history:eceived 19 November 2014eceived in revised form 30 April 2015ccepted 1 May 2015vailable online xxx

eywords:ypoxiaypercapniaypercapnic hypoxia

a b s t r a c t

Respiratory responses to hypoxia and/or hypercapnia, and their relationship to neural activity in the ven-trolateral medulla (VLM), which includes the respiratory center, have not yet been elucidated in detail.We herein examined respiratory responses during exposure of 10% O2 (hypoxia), 10% CO2 (hypercapnia),and 10% O2–10% CO2 (hypercapnic hypoxia) using plethysmography. In addition to recording respira-tion, Fos expressions were examined in the VLM of the rat exposed to each gas to analyze neural activity.Respiratory frequency was increased in rats exposed to hypoxia, and Fos-positive neurons were observedin the caudal VLM (cVLM) and medial VLM (mVLM). Tidal volume was increased in rats exposed to hyper-capnia, and Fos-positive neurons were observed in the rostral VLM (rVLM) includes the retrotrapezoidnucleus (RTN) and mVLM. Tidal volume was enhanced in rats exposed to hypercapnic hypoxia, similar

entrolateral medullahemoreceptionespiratory response

to that in hypercapnia-exposed rats, and Fos-positive neurons were observed in the entire region of theVLM. In the mVLM and cVLM, double immunofluorescence showed Fos-immunoreactive nerve cells werealso immunoreactive to dopamine �-hydroxylase, the marker for A1/C1 catecholaminergic neuron. Theseresults suggested that hypoxia and hypercapnia modulated rhythmogenic microcircuits in the mVLM viaA1/C1 neurons and the RTN, respectively.

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. Introduction

Respiration rhythm is known to be affected by hypoxic orypercapnic exposure (Marczak et al., 2004; Smith et al., 2006).

Please cite this article in press as: Wakai, J., et al., Differenctrolateral medulla of rats exposed to hypoxia, hypercapnia, anhttp://dx.doi.org/10.1016/j.resp.2015.05.008

ew studies have compared hypoxic exposure to hypercapnicxposure in rats. Holley et al. (2012) reported that hypoxic expo-ure increased respiratory frequency while hypercapnic exposure

Abbreviations: AMB, nucleus ambiguus; BC, Bötzinger complex; cVLM, cau-al ventrolateral medulla; DBH, dopamine �-hydroxylase; DRG, dorsal respiratoryroup; IO, inferior olive; KF, Kölliker-Fuse nucleus; LRN, lateral reticular nucleus;VLM, medial ventrolateral medulla; NTS, nucleus of the solitary tract; PBC,

re-Bötzinger complex; PGRN, paragigantocellular reticular nucleus; RTN, retro-rapezoid nucleus; rVLM, rostral ventrolateral medulla; SO, superior olive nucleus;II, facial nucleus; VLM, ventrolateral medulla; VRC, ventral respiratory column;RG, ventral respiratory group.∗ Corresponding author at: Laboratory of Veterinary Anatomy and Cell Biology,aculty of Agriculture, Iwate University, 18-8, Ueda 3-chome, Morioka, Iwate 020-550, Japan. Tel.: +81 19 621 6273; fax: +81 19 621 6273.

E-mail address: [email protected] (Y. Yamamoto).

ttp://dx.doi.org/10.1016/j.resp.2015.05.008569-9048/© 2015 Published by Elsevier B.V.

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© 2015 Published by Elsevier B.V.

increased both respiratory frequency and tidal volume. On theother hand, Walker et al. (1985) reported that exposure to bothhypoxia and hypercapnia increased respiratory frequency and tidalvolume. Moreover, increases induced in respiratory frequency bylow PaO2 were inhibited when PaCO2 was elevated in rats exposedto hypercapnic hypoxia (Cragg and Drysdale, 1983). Thus, thesefindings suggest that a relationship exists between hypoxia andhypercapnia in respiratory responses.

Respiratory responses to hypoxia are evoked by the carotidbody, which is a peripheral chemoreceptor. Chemosensitive cellsin the carotid body are excited by decreases in PaO2, and signalsreach respiratory centers in the medulla oblongata and pons viathe nucleus of the solitary tract (NTS; Peers, 1997; Prabhakar, 2006).Regarding CO2 and acidity, the main sensor for changes in CO2/pHis considered to be the retrotrapezoid nucleus (RTN) in the medullaoblongata (Guyenet et al., 2009, 2010); however, the carotid body

es in respiratory changes and Fos expression in the ven-d hypercapnic hypoxia. Respir. Physiol. Neurobiol. (2015),

is also sensitive to decreases in blood pH by hypercapnic exposure(Gonzalez et al., 1994). Sensory information from peripheral andcentral chemosensory organs/areas is transmitted to respiratorycenters; the dorsal respiratory group (DRG) and ventral respiratory

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mailto:[email protected]

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Fig. 1. Schematic illustration of the method for head-out plethysmograph.A respiration waveform was obtained by detecting the inflow and outflow of air inthe plethysmograph-chamber using a flow head and spirometer pod connected toPowerLab. When gasses flowed from the inlet, the head of animal was exposed to

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ARTICLEESPNB 2499 1–9

J. Wakai et al. / Respiratory Physio

olumn (VRC) in the medulla oblongata as well as the pons respi-atory group (PRG).

Of these respiratory centers, the VRC has been shown to play key role in autonomic respiratory control (Ott et al., 2011). TheRC is located in the ventrolateral medulla (VLM) and contains theTN, Bötzinger complex (BC), pre-Bötzinger complex (PBC), rostralentral respiratory group (rVRG), and caudal ventral respiratoryroup (cVRG; Feldman and McCrimmon, 2008). The PBC containst least four kinds of inspiratory and expiratory neurons consist-ng of rhythm-generating microcircuits, and the BC transmits thehythms generated to lower neurons (Smith et al., 1991, 2007; Sunt al., 1998; Koshiya and Smith, 1999). The rVRG and cVRG alsoontain interneurons leading to the rostral medulla oblongata andulbospinal respiratory premotor neurons for the control of motoreurons (Ellenberger and Feldman, 1990b; Ellenberger et al., 1990;alia, 1981).

In addition to the VRC, noradrenergic A1 neurons and adren-rgic C1 neurons are also involved in respiratory control in theedulla oblongata. A1/C1 neurons are located ventral to the VRC,

nd some A1/C1 neurons are intermingled with neurons in the BCnd rVRG/cVRG, respectively (Ellenberger et al., 1990). Previoustudies reported that A1/C1 neurons were activated by hypoxicxposure because Fos-positive cells were observed in the A1/C1eurons of rats exposed to hypoxia (Erickson and Millhorn, 1994;mith et al., 1995). These findings indicated that A1/C1 neurons mayontribute to respiratory responses to changes in environmentalas.

Although respiratory changes and the regulatory system inhe central circuit for respiratory regulation have not yet beenxamined in detail under hypoxic and hypercapnic exposure, thectivities of the VLM have been suggested to be modulated by gasxposure. In the present study, we examined respiratory responseso hypoxia (10% O2), hypercapnia (10% CO2), and hypercapnicypoxia (10% O2–10% CO2) using plethysmography. The topograph-

cal expression pattern of Fos, a marker of neuronal activity, wasetermined in the respiratory centers of rats exposed to these gasesor 2 h. We focused on the interrelationship between respiratoryesponses and Fos expression in the VLM in order to elucidate theeural mechanisms underlying hypoxia- and hypercapnia-evokedespiratory responses.

. Materials and methods

Animal experimental protocols were approved by the Commit-ee on the Use of Live Animals in Teaching and Research of Iwateniversity (approval number: A201047).

.1. Head-out plethysmography

.1.1. Gas exposure experimentsMale Wistar rats (8 weeks old) were divided into four exper-

mental groups (n = 4 in each group) were exposed to differentases: 1) hypoxic group (10% O2 balanced N2), 2) hypercap-ic group (20% O2 and 10% CO2 balanced N2), 3) hypercapnicypoxic group (10% O2 and 10% CO2 balanced N2), and 4) con-rol group (20% O2 balanced N2). These gas exposure conditionsere decided by reference to previously studies (Wakai et al., 2010;hannouchi et al., 2013; Takakura and Moreira, 2013; Lemes andoccal, 2014; Damasceno et al., 2015). Each rat was anesthetizedith urethane (1.5 g/kg; intraperitoneal injection) and placed into

head-out plethysmograph-chamber inside an acrylic chamber


50 × 40 × 50 cm). Three holes, located at the top of a sidewall ofhe acrylic chamber, were connected to three types of gas bombsO2, CO2, and N2). O2 and CO2 levels within the chamber were

onitored with a gas analyzer and each gas level was maintained

the gas. Gas concentration in the acrylic chamber was adjusted by N2, CO2 and/orO2 gasses that automatically regulated by solenoid valve (V) connected with O2 andCO2 sensors.

automatically at a constant level. In the hypoxic experiment, O2concentrations were maintained at 9.8–10.2% by N2 influx. In thehypercapnic experiment, CO2 and O2 concentrations were main-tained at 9.6–10.4% by CO2 influx and at 20–20.5% by O2 influx,respectively. In the hypercapnic hypoxic experiment, O2 and CO2concentrations were maintained at 9.6–10.4% by N2 influx and at9.6–10.4% by CO2 influx, respectively. In the control experiment, O2concentrations were maintained at 20–20.5% by O2 influx and CO2concentrations were maintained at <1% by N2 influx. The temper-ature within the chamber was maintained at 25 ◦C.

2.1.2. Measurement of respiration in rats exposed to each gasRespiration measurements were collected in all rats for 30 min

before gas exposure. Gas exposure experiments and respirationmeasurements were examined for 60 min. We measured changesin the respiration of rats by detecting changes in the internal pres-sure of the plethysmograph-chamber using a respiratory flow head(MLA 1L, ADInstruments, Sydney, Australia) and spirometer pod(ML311, ADInstruments) connected to PowerLab (ADInstruments).The experimental equipment for head-out plethysmograph is illus-trated in Fig. 1. Respiration data were analyzed by LabChart 7(ADInstruments). Flow volume was calibrated according to themanufacturer’s protocol. In order to obtain a respiration waveformfor respiratory volume, we integrated a waveform of changes inthe internal pressure of the plethysmograph chamber. Respiratoryparameters obtained by plethysmography have high correlationwith the parameters obtained by pneumotachography in mice orrats (Onodera et al., 1997; Nirogi et al., 2012; Hoymann, 2012).Therefore, we considered the respiratory volume obtained in thepresent study was comparable to tidal volume. We examined respi-ratory frequencies, tidal volumes, and respiratory minute volumesfor 1 min before and at five time points after (0, 15, 30, 45 and 60 minafter exposure to each gas) from the waveforms of respiratory vol-ume.

2.2. Statistical analysis

Statistical analyses were performed using the Kruskal–Wallistest with post hoc test (Games–Howel test) with p < 0.05 beingconsidered significant.

2.3. Immunohistochemistry


2.3.1. Fos-immunohistochemistryMale Wistar rats (8 weeks old) were divided into four experi-

mental groups that were the same as those described previously(n = 6/each group). Each rat was exposed to each gas for 2 h at

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Fig. 2. Location of the rVLM, mVLM, and cVLM.This figure shows a sagittal section of medulla oblongata. In this study, we observedthe rVLM, mVLM, and cVLM. The rVLM is located ventral to the facial nucleus. ThemVLM is located caudal to the facial nucleus and ventral to the AMB. The mVLMis located caudal to the mVLM and dorsal to the LRN. SO: Superior olive, VII: facialntm

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ucleus, AMB: nucleus ambiguus, LRN: lateral reticular nucleus, rVLM: rostral ven-rolateral medulla, mVLM: medial ventrolateral medulla, cVLM: caudal ventrolateral

edulla

5 ◦C in a cage (16 × 27 × 13 cm; free-feeding) inside the acrylichamber. After the exposure experiments, rats were anesthetizedith pentobarbital sodium (50 mg/kg; intraperitoneal injection)

nd perfused transcardially with Ringer’s solution (200 mL) fol-owed by Zamboni’s fixative (4% paraformaldehyde, 0.5% picric acidn 0.1 M phosphate buffer; pH 7.4). The brains were removed andmmersed in the same fixative overnight at 4 ◦C. They were theninsed (3× 10 min) in phosphate buffered saline (PBS; pH 7.4),oaked in 30% sucrose in PBS, and frozen. Serial transverse sec-ions (50 �m) were cut on the cryostat. The sections were collectedlternatively and divided two series. One series of sections wasrocessed for Nissl stain while the other was used for immuno-istochemistry (avidin-biotin-peroxidase complex method; ABCethod). Nissl stain sections were used to identify medullary

uclei.Regarding enzyme immunohistochemistry, free-floating sec-

ions were first rinsed in 0.5% Triton X-100 in PBS overnight at 4 ◦C.n order to block non-specific peroxidase activity, the sections werencubated in PBS containing 1.5% H2O2 for 1 h at room temperaturend then rinsed in PBS (3× 10 min). The sections were then incu-ated for 72 h at 4 ◦C with rabbit polyclonal antiserum against FosAb-5, 1:10,000, Oncogene Research Products, Cambridge, MA) andon-immune donkey serum (1:50) to prevent non-specific bind-

ng sites. After being incubated, the sections were rinsed in PBS3× 20 min) and incubated for 1 h in biotinylated anti-rabbit IgGonkey serum (1:1000, Jackson ImmunoResearch Products, Westrove, PA) and washed in PBS (3× 20 min). The sections were

hen incubated in an avidin-biotin-peroxidase complex (Vectastainlite ABC kit; Vector Laboratories, Burlingame, CA) for 2 h. Afterashing with PBS (3× 20 min), sections were incubated in 0.02%

,3′-diaminobenzidine tetrahydrochloride in Tris–HCl buffer solu-ion (pH 7.4) in the presence of 0.0006% H2O2 for 15–30 min atoom temperature. Finally, after two washes in PBS, sections wereounted on gelatin-coated slides, air-dried in an incubator at 37 ◦C,


ehydrated in alcohol, cleared in xylene, and coverslipped.For observation, the ventrolateral medulla was conveniently

ivided into three parts, i.e., the rostral, medial, and caudal partsrVLM, mVLM, cVLM; Fig. 2). The rVLM is located ventral to the

PRESS Neurobiology xxx (2015) xxx–xxx 3

facial nucleus and ventrolateral to the paragigantocellular reticu-lar nucleus lateral part (PGRN). The mVLM is a part of the PGRNand is located caudal to the facial nucleus, rostral to the LRN, andventral to the nucleus ambiguus (AMB). The cVLM is located cau-dal to the LRN and regions that enclose the LRN and AMB. Guyenetand Wang (2001), Stornetta et al. (2009), and Wang et al. (2001)reported that the rVLM includes the RTN, the mVLM includes theBC and PBC, and the cVLM includes the rVRG and cVRG. The num-bers of positive neurons were counted in all sections stained forFos-immunohistochemistry within the range of rVLM, mVLM orcVLM. The cell counting was performed bilaterally on each sectionunder a light microscope. Statistical analyses were performed usingthe Kruskal–Wallis test with post hoc test (Games-Howel test) withp < 0.05 being considered significant.

2.3.2. Double immunofluorescence for Fos and DBHSome neurons belonging to noradrenergic A1 and adrenergic

C1 groups were previously shown to be intermingled with neu-rons of the mVLM and cVLM (Ellenberger et al., 1990). To clarifywhether Fos-positive neurons were catecholaminergic neurons ornon-catecholaminergic neurons, we performed double staining forFos and dopamine �-hydroxylase (DBH), which is an indicator ofnoradrenergic or adrenergic neurons. For indirect immunofluores-cence, serial transverse sections (20 �m) from additional animalsof each group were cut on the cryostat, mounted on slides, andair-dried for 1–2 h. Sections were rinsed in PBS (3× 5 min) andincubated for 24 h at 4 ◦C with rabbit polyclonal antiserum againstFos (Ab-5, 1:5000, Oncogene Research Products, Cambridge, MA),mouse monoclonal antiserum against DBH (MAB308, ChemiconInternational, Temecula, CA), and non-immune donkey serum(1:40). After being incubated, sections were rinsed in PBS (3×10 min), incubated for 2 h in Alexa 488-conjugated donkey anti-rabbit IgG (1:200, Jackson ImmunoResearch Products, West Grove,PA) and Cy3-conjugated donkey anti-mouse IgG (1:200, JacksonImmunoResearch Products, West Grove, PA), and then washedin PBS (3× 20 min). Sections were then coverslipped with Flu-oromount (Diagnostic BioSystems, Pleasanton, CA). The sectionsstained by double immunofluorescence were observed under aconfocal laser microscope (C2, Nikon, Tokyo). All images were ana-lyzed with the use of Photoshop CS5 (Adobe Systems, San Jose, CA)and NIS-Elements (Nikon, Tokyo).

3. Results

3.1. Measurement of respiration using head-outplethysmography

The representative waveform of flow and volume derived fromplethysmography 30 min after gas exposure is shown in Fig. 3.Table 1 shows tidal volumes, respiratory frequencies, and respira-tory minute volumes at each time point in rats exposed to hypoxia,hypercapnia, hypercapnic hypoxia, and control gas. When rats wereexposed to hypoxia, respiratory frequency was significantly higherat 15 min and 30 min, while respiratory minute volume was signifi-cantly higher at 15 min than at 0 min. Tidal volumes and respiratoryminute volumes in rats exposed to hypercapnia and hypercapnichypoxia were significantly higher at 15, 30, 45 and 60 min than at0 min.

Respiratory minute volume, tidal volume, and respiratory fre-quency values at 0 min were set to 100%, and compared with eachvalue every 15 min. Respiratory minute volume was enhanced to


approximately 130, 300, and 250% in rats exposed to hypoxia,hypercapnia, and hypercapnic hypoxia, respectively (Fig. 4A).Respiratory minute volume was higher in rats exposed to hypoxiathan in those in the control group at 15, 30 and 45 min. Greater

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Fig. 3. Respiration waveform of changes in flow and volume 30 min after exposure to each gas.I een prr hile f

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n rats exposed to hypoxia, the amplitude of both flow and volume are similar betwats exposed to hypercapnia and hypercapnic hypoxia, the amplitude is increased w

hanges were induced in respiratory minute volume by hypercap-ia or hypercapnic hypoxia than by hypoxia or the control gast all the time points examined. Tidal volume was enhanced topproximately 270 and 250% in rats exposed to hypercapnia and


ypercapnic hypoxia, respectively, but was not enhanced in thosexposed to hypoxia (Fig. 4B). Tidal volume was significantly highern rats exposed to hypercapnia or hypercapnic hypoxia than in ratsxposed to control gas in all the time points examined. On the

able 1hanges of tidal volume, respiration frequency and respiratory minute volume during ex

0 min 15 min

Tidal volume (mL)Hypoxia 0.37 ± 0.031 0.35 ± 0.031

Hypercapnia 0.40 ± 0.047 1.03 ± 0.166*

Hypercapnic hypoxia 0.52 ± 0.035 1.22 ± 0.098*

Control 0.45 ± 0.038 0.44 ± 0.058

Respiratory frequency (breath/min)Hypoxia 125.8 ± 17.5 183.0 ± 20.0*

Hypercapnia 131.5 ± 9.3 138.5 ± 5.9

Hypercapnic hypoxia 120.8 ± 9.1 139.3 ± 4.6

Control 114.5 ± 23.4 108.0 ± 17.6

Respiratory minute volume (mL/min)Hypoxia 46.6 ± 3.9 63.0 ± 8.1*

Hypercapnia 51.8 ± 6.6 142.0 ± 16.3*

Hypercapnic hypoxia 62.5 ± 3.6 169.3 ± 8.0*

Control 51.1 ± 10.5 47.2 ± 8.8

ypoxia, 10% O2; Hypercapnia, 10% CO2; Hypercapnic hypoxia, 10% O2–10% CO2.verage ± S.D.* Significant vs 0 min (pre-exposure term) at p < 0.05.

e-exposure and 30 min after exposure, and respiration is increased (upper row). Inrequency remains unchanged (middle and lower rows).

other hand, no significant difference was observed in tidal volumebetween the hypoxic and control groups. Respiratory frequencywas enhanced to 130–150% in hypoxic rats and was significantlyhigher than that in the control and hypercapnic groups at 15 min,


the control, hypercapnic and hypercapnic hypoxic groups at 30 min,and hypercapnic group at 45 min. Respiratory frequency was notenhanced in rats exposed to hypercapnia or hypercapnic hypoxia(Fig. 4C).

posure of hypoxia, hypercapnia and hypercapnic hypoxia.

30 min 45 min 60 min

0.34 ± 0.03 0.35 ± 0.03 0.34 ± 0.021.11 ± 0.10* 1.12 ± 0.11* 1.10 ± 0.09*

1.31 ± 0.16* 1.32 ± 0.18* 1.31 ± 0.21*

0.44 ± 0.09 0.41 ± 0.08 0.41 ± 0.07

184.5 ± 19.2* 167.3 ± 16.8 167.8 ± 26.2137.0 ± 7.8 137.3 ± 5.4 140.1 ± 10.7124.5 ± 12.6 122.5 ± 16.1 123.0 ± 22.7110.8 ± 18.5 115.0 ± 28.8 119.5 ± 10.5

62.2 ± 7.9 58.8 ± 7.6 57.4 ± 8.3152.0 ± 7.4* 153.5 ± 11.6* 154.0 ± 13.9*

161.3 ± 4.4* 159.0 ± 6.2* 157.5 ± 5.8*

47.7 ± 8.9 45.8 ± 8.7 45.2 ± 8.0

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Please cite this article in press as: Wakai, J., et al., Differences in respiratory changes and Fos expression in the ven-trolateral medulla of rats exposed to hypoxia, hypercapnia, and hypercapnic hypoxia. Respir. Physiol. Neurobiol. (2015),http://dx.doi.org/10.1016/j.resp.2015.05.008


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Fig. 4. Changes in tidal volume (VT), respiratory frequency (Rf), and respiratory minute volume (VE) expressed as a percentage.The values of respiratory minute volume, tidal volume, and respiratory frequency at 0 min were set to 100%, and compared with each value every 15 min. Panels A, B,and C show changes in tidal volume, respiratory frequency, and respiratory minute volume in the four experimental groups, respectively. In rats exposed to hypercapniaand hypercapnic hypoxia, respiratory minute volume and tidal volume are enhanced to 200% at all the exposure times examined, whereas respiratory frequency remainsunchanged. In rats exposed to hypoxia, respiratory minute volume and respiratory frequency are significantly increased at 15, 30 and 45 min and tidal volume remainsunchanged. *1: p < 0.05 vs the hypoxic and control groups, *2: p < 0.05 vs hypercapnic and control groups, *3: p < 0.05 vs the hypercapnic, hypercapnic hypoxic, and controlgroups, *4: p < 0.05 vs the hypercapnic group, *5: p < 0.05 vs control group.

Fig. 5. Fos immunoreactivity in the rVLM.A few Fos-positive neurons are found in sections from rats exposed to hypoxia (A). In rats exposed to hypercapnia (B) and hypercapnic hypoxia (C), Fos-positive neurons areexpressed in the RTN ventral to the facial nucleus (VII). Few Fos-positive neurons appear in the rVLM region of control rats.

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ig. 6. Fos immunoreactivity in the mVLM.os-positive neurons are widely distributed in the PGRN of rats exposed to hypoxia (n control rats (D).

.2. Fos-positive neurons in the VLM

Few Fos-positive neurons were detected in the surface region


n hypoxia exposed and control rats, and the numbers of posi-ive neurons were 9.7 ± 4.0 and 7.0 ± 6.4, respectively (Fig. 5A, D).umerous Fos-positive neurons were detected in the rVLM of ratsxposed to hypercapnia and hypercapnic hypoxia, and positive

ig. 7. Fos immunoreactivity in the cVLM.os-positive neurons are observed in the PGRN surrounded by the AMB and LRN in sectioeurons appear in sections from rats exposed to hypercapnia (B) and control rats (D).

ercapnia (B), and hypercapnic hypoxia (C). Fos-positive neurons were not observed

neurons accounted for 317.8 ± 25.6 and 363.2 ± 25.6 per area,respectively (Fig. 5B, C). Fos-positive neurons were also observedin the RTN ventral to the facial nucleus including the ventral sur-


face of the medulla. The numbers of Fos-positive neurons weresignificantly higher in rats exposed to hypercapnia and hypercap-nic hypoxia than in rats exposed to hypoxia or the control gas(Fig. 8A). No significant differences were observed between the

ns from rats exposed to hypoxia (A) and hypercapnic hypoxia (C). Few Fos-positive

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Fig. 8. Number of Fos-positive neurons in the rVLM (A), mVLM (B), and cVLM (C).The numbers of Fos-positive cells in the rVLM are significant higher with hyper-capnia and hypercapnic hypoxia than with hypoxia and control gas. In the mVLM,the number of Fos-positive cells is significantly higher in the experimental groupsfor gas exposure than in the control. In the cVLM, the number of Fos-positive cellsis significantly higher in the hypoxic group than in the other groups, and is higherin the hypercapnic hypoxia group than in the hypercapnia and control groups. 1:

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ypercapnic group and hypercapnic hypoxic group or between theypoxic group and control group.

Fos-positive neurons were detected in the mVLM of rats exposedo hypoxia (207.0 ± 32.1 per area), hypercapnia (297.7 ± 73.5), andypercapnic hypoxia (296.2 ± 64.7) (Fig. 6A–C). Only a few Fos-ositive neurons were observed in the mVLM of the control group19.8 ± 18.0) (Fig. 6D). In rats exposed to hypoxia, Fos-positive neu-ons were concentrated in the ventral part, while those in ratsxposed to hypercapnia or hypercapnic hypoxia broadly extendedhroughout the mVLM. The mean numbers of Fos-positive neuronsn the mVLM were significantly higher in the three experimentalroups than in the control group (Fig. 8B). The number of Fos-ositive neurons was significantly lower in hypoxic rats than inats exposed to hypercapnia and hypercapnic hypoxia.

Many Fos-positive neurons were observed around the AMBn the cVLM of rats exposed to hypoxia or hypercapnic hypoxiaFig. 7A, C). Few positive neurons were detected in the hypercapnicr control group (Fig. 7B, D). As shown in Fig. 8C, the number of Fos-ositive neurons was significantly higher in rats exposed to hypoxia364.8 ± 69.5 per area) or hypercapnic hypoxia (187.5 ± 81.2) thann hypercapnic (55.3 ± 36.4) or control rats (13.3 ± 12.9). Further-

ore, a significant difference was noted between hypoxia andypercapnic hypoxia, but not between hypercapnia and the control.

.3. DBH immunoreactivity in Fos-positive neurons in the VLM

Fos-positive neurons in the rVLM of rats exposed to hypercapniand hypercapnic hypoxia did not show immunoreactivity for DBHFig. 9A). Some Fos-positive neurons in the mVLM of all groupsere also immunoreactive for DBH; however, Fos single positiveeurons were also observed (Fig. 9B). In the cVLM of rats exposedo hypoxia or hypercapnic hypoxia, most Fos-positive neurons werelso positive for DBH (Fig. 9C, D). A few Fos-positive neurons didot show immunoreactivity for DBH.

. Discussion

.1. Methodological considerations

In the present study, measurement of respiration in the ratxposed to each gas was performed under urethane anesthesia.hile urethane anesthesia is known its minimal effects on car-

iovascular and respiratory systems and maintenance of spinaleflexes (Maggi and Meli, 1986; Hara and Hariis, 2002), ani-als under urethane anesthesia are used as a model of sleep

Pagliardini et al., 2013). Respiration pattern of urethane anes-hetized animals show similar to that of non-REM sleep animals,.e., decrease of minute ventilation and respiratory frequency isecreased (Pagliardini et al., 2013). Thus, it may be the respirationatterns are more or less different between urethane-anesthetizednd conscious animal. In the present study, respiratory responses toypoxia, hypercapnia or hypercapnic hypoxia were distinctly dif-

erent. On the other hand, immunohistochemical analysis showedxperiment-specific distribution of Fos-immunoreactive nerve cellodies in the rat exposed to hypoxia, hypercapnia and hypercapnicypoxia in comparison to control. Thus, we consider that both phys-

ological and immunohistochemical analyses would be representhe effect of three different environmental gasses.

.1.1. Localization of Fos-positive neurons in rVLMPrevious studies observed Fos-positive neurons in the RTN

f mice and cats exposed to hypercapnia (Niblock et al., 2012;


eppema et al., 1994). In the present study, Fos-positive neuronsere observed in the rVLM including the RTN of rats exposed toypercapnia or hypercapnic hypoxia, but not in rats exposed toypoxia. CO2/H+ sensitive neurons have been identified in the RTN

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p < 0.05 vs hypoxic group, 2: p < 0.05 vs hypercapnic group, 3: p < 0.05 vs hypercapnichypoxic group, 4: p < 0.05 vs control.

using patch clamp techniques (Lazarenko et al., 2009). Moreover,bilateral destruction of the RTN was shown to inhibit respira-tory responses to hypercapnia (Akilesh et al., 1997). The presentresults are in good agreement with these reports. On the otherhand, although the firing rate of RTN neurons was reported to beincreased by the carotid body with hypoxia (Takakura et al., 2006),RTN was not activated in the present study. Thus, RTN neuronsmay not play significant roles in respiratory regulation for hypoxicexposure.

RTN neurons were found to project to the mVLM, espe-cially the BC and PBC, by the anterograde tracer biotinylateddextran amine, and a large part of these projection neurons con-tained vesicular glutamate transporter 2 and galanin (Rosin et al.,


2006; Bochorishvili et al., 2012). Since the BC and PBC containrhythmogenic microcircuits for respiration (Smith et al., 2013),excitatory neurotransmitters such as glutamate in the RTN may

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Fig. 9. Double immunofluorescence for Fos and DBH in the VLM.( are nD showna re also

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A) The rVLM of rats exposed to hypercapnia. Fos-positive neurons (green) in the RTNouble positive neurons (arrows) and neurons positive for Fos, but not for DBH are

re concentrated. Some neurons that immunoreacted with Fos, but not with DBH a

ffect respiratory rhythms to regulate respiratory minute volumend tidal volume under hypercapnic conditions.

.1.2. Localization of Fos-positive neurons in the mVLMA close relationship has been reported between the activity of

he phrenic nerve and neurons in the mVLM or cVLM (Smith et al.,000). The PBC displays autonomic firing prior to inspiration, andhe BC contributes to the switch from inspiration to expirationEzure et al., 2003; Shen et al., 2003; Smith et al., 2007). Further-

ore, the mVLM contains several distinct respiratory neuron typesncluding excitatory and inhibitory neurons to make microcircuitsor rhythmogenesis (Schwarzacher et al., 1991; Shen et al., 2013;mith et al., 2013).

A previous study demonstrated that the administration of theABAB receptor agonist baclofen to the mVLM induced an increase

n the respiratory frequency of rabbits (Bongianni et al., 2010). Theespiratory frequency of rats exposed to hypoxia was increased inhe present study; therefore, Fos-positive neurons in the mVLM

ay contain GABAergic inhibitory neurons. On the other hand, thedministration of glutamate to the PBC has been shown to inducen increase in the respiratory minute volume of rats (Moraes et al.,011). In rats exposed to hypercapnia and hypercapnic hypoxia,os-positive neurons in the mVLM may also contain excitatory glu-amatergic neurons because respiratory minute volume and tidalolume were elevated during gas exposure. The smaller numberf Fos-positive neurons in hypoxic rats suggests that excitatoryeurons are limited. Furthermore, the disruption of neurotransmis-ion in the mVLM by the administration of colchicine was reportedo inhibit the increases induced in tidal volume, respiratory fre-uency, and respiratory minute volume by an exposure to hypoxiar hypercapnia (Wu et al., 2005). Our results suggest that the mVLMlays a key role in respiratory modulation by both hypoxia andypercapnia.


.1.3. Localization of Fos-positive neurons in the cVLMIn the cVLM, excitatory premotor neurons exist between rhyth-

ogenic microcircuits in the BC and PBC (Smith et al., 2013).

ot immunoreactive for DBH. (B) The mVLM of rats exposed to hypercapnic hypoxia. (arrowhead). (C, D) The cVLM of rats exposed to hypoxia. Double positive neurons

shown (arrowhead). (F) is a higher magnification view of (E).

The rostral VRG and caudal VRG in the cVLM contain bulbospinalpremotor neurons, and the premotor neurons project to inspira-tory motor neurons and expiratory motor neurons, respectively(Smith et al., 2013). In addition to the premotor neurons in theVRG, catecholaminergic A1/C1 neurons have been detected alongthe VRG (review see Guyenet et al., 2013). In the present study,most Fos-positive neurons in the cVLM may be A1/C1 neuronsbecause they showed DBH immunoreactivity. The presence ofFos-positive neurons in rats exposed to hypoxia and hypercap-nic hypoxia suggests that A1/C1 neurons were activated by thehypoxic stimulation. The present results are consistent with pre-vious findings published by Smith et al. (1995), in which Fosimmunoreactivity was observed in A1/C1 neurons in the cVLM ofrats exposed to hypoxia. Furthermore, few Fos-positive neuronswere observed in rats exposed to hypercapnia, which indicates thatthe activation of A1/C1 neurons may specific to hypoxia, not tohypercapnia. A previous study reported that A1/C1 neurons exhib-ited Fos immunoreactivity following an electrical stimulus to thecarotid sinus nerve, which mimicked hypoxic exposure (Ericksonand Millhorn, 1994). Moreover, projections to the cVLM from theNTS, which is the receiving input of the carotid sinus nerve, wereidentified in tracer experiments (Aicher et al., 1995). Therefore,Fos-positive A1/C1 neurons in the cVLM may mediate inputs fromthe carotid body when animals are exposed to hypoxia, but notto hypercapnia. On the other hand, retrograde tracer experimentsshowed projections to the mVLM from the cVLM (Ellenberger andFeldman, 1990a). Moreover, the administration of the adrenaline�2 receptor antagonist, yohimbine, to the mVLM decreased the fir-ing rate of the phrenic nerve in a medullary preparation of mice inwhich the pons and dorsal medulla were removed (Zanella et al.,2006). Based on these findings, the nerve endings of A1/C1 neuronsin the mVLM may release catecholamine to enhance respiratoryfrequency.


In conclusion, we speculate that hypoxia and hypercap-nia modulated rhythmogenic microcircuits in the mVLM viaA1/C1 neurons in the cVLM and the RTN in the rVLM,respectively.

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