Differences in Respiratory Changes... - Apres. Silvia Zebral

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Respiratory Physiology & Neurobiology xxx (2015) xxxxxx
Contents lists available at ScienceDirect
Respiratory Physiology & Neurobiology

journal homepage: www.elsevier.com/locate/resphysiol
Differences in respiratory changes and Fos expression in the

ventrolateral medulla of rats exposed to hypoxia, hypercapnia, and
hypercapnic hypoxia
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Jun Wakai a , Daichi Takamura b , Ryosuke Morinaga c , Nobuaki Nakamuta c ,

Yoshio Yamamoto c,
a
Laboratory Animal Research Center, Fukushima Medical University School of Medicine, Fukushima, Japan
Laboratory of Veterinary Biochemistry and Cell Biology, Faculty of Agriculture, Iwate University, Morioka, Japan
c
Laboratory of Veterinary Anatomy and Cell Biology, Faculty of Agriculture, Iwate University, Morioka, Japan
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a r t i c l e
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a b s t r a c t
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Article history:
Received 19 November 2014
Received in revised form 30 April 2015
Accepted 1 May 2015
Available online xxx
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Keywords:
Hypoxia
Hypercapnia
Hypercapnic hypoxia
Ventrolateral medulla
Chemoreception
Respiratory response
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1. Introduction
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Respiratory responses to hypoxia and/or hypercapnia, and their relationship to neural activity in the ventrolateral 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 respiration, 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 observed
in the caudal VLM (cVLM) and medial VLM (mVLM). Tidal volume was increased in rats exposed to hypercapnia, and Fos-positive neurons were observed in the rostral VLM (rVLM) includes the retrotrapezoid
nucleus (RTN) and mVLM. Tidal volume was enhanced in rats exposed to hypercapnic hypoxia, similar
to that in hypercapnia-exposed rats, and Fos-positive neurons were observed in the entire region of the
VLM. In the mVLM and cVLM, double immunouorescence showed Fos-immunoreactive nerve cells were
also immunoreactive to dopamine -hydroxylase, the marker for A1/C1 catecholaminergic neuron. These
results suggested that hypoxia and hypercapnia modulated rhythmogenic microcircuits in the mVLM via
A1/C1 neurons and the RTN, respectively.
2015 Published by Elsevier B.V.
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Respiration rhythm is known to be affected by hypoxic or

hypercapnic exposure (Marczak et al., 2004; Smith et al., 2006).
Few studies have compared hypoxic exposure to hypercapnic
exposure in rats. Holley et al. (2012) reported that hypoxic exposure increased respiratory frequency while hypercapnic exposure
Abbreviations: AMB, nucleus ambiguus; BC, Btzinger complex; cVLM, caudal ventrolateral medulla; DBH, dopamine -hydroxylase; DRG, dorsal respiratory
group; IO, inferior olive; KF, Klliker-Fuse nucleus; LRN, lateral reticular nucleus;
mVLM, medial ventrolateral medulla; NTS, nucleus of the solitary tract; PBC,
pre-Btzinger complex; PGRN, paragigantocellular reticular nucleus; RTN, retrotrapezoid nucleus; rVLM, rostral ventrolateral medulla; SO, superior olive nucleus;
VII, facial nucleus; VLM, ventrolateral medulla; VRC, ventral respiratory column;
VRG, ventral respiratory group.
Corresponding author at: Laboratory of Veterinary Anatomy and Cell Biology,
Faculty of Agriculture, Iwate University, 18-8, Ueda 3-chome, Morioka, Iwate 0208550, Japan. Tel.: +81 19 621 6273; fax: +81 19 621 6273.
E-mail address: yyoshio@iwate-u.ac.jp (Y. Yamamoto).
increased both respiratory frequency and tidal volume. On the

other hand, Walker et al. (1985) reported that exposure to both
hypoxia and hypercapnia increased respiratory frequency and tidal
volume. Moreover, increases induced in respiratory frequency by
low PaO2 were inhibited when PaCO2 was elevated in rats exposed
to hypercapnic hypoxia (Cragg and Drysdale, 1983). Thus, these
ndings suggest that a relationship exists between hypoxia and
hypercapnia in respiratory responses.
Respiratory responses to hypoxia are evoked by the carotid
body, which is a peripheral chemoreceptor. Chemosensitive cells
in the carotid body are excited by decreases in PaO2 , and signals
reach respiratory centers in the medulla oblongata and pons via
the nucleus of the solitary tract (NTS; Peers, 1997; Prabhakar, 2006).
Regarding CO2 and acidity, the main sensor for changes in CO2 /pH
is considered to be the retrotrapezoid nucleus (RTN) in the medulla
oblongata (Guyenet et al., 2009, 2010); however, the carotid body
is also sensitive to decreases in blood pH by hypercapnic exposure
(Gonzalez et al., 1994). Sensory information from peripheral and
central chemosensory organs/areas is transmitted to respiratory
centers; the dorsal respiratory group (DRG) and ventral respiratory
http://dx.doi.org/10.1016/j.resp.2015.05.008
1569-9048/ 2015 Published by Elsevier B.V.
Please cite this article in press as: Wakai, J., et al., Differences in respiratory changes and Fos expression in the ventrolateral medulla of rats exposed to hypoxia, hypercapnia, and hypercapnic hypoxia. Respir. Physiol. Neurobiol. (2015),
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column (VRC) in the medulla oblongata as well as the pons respiratory group (PRG).
Of these respiratory centers, the VRC has been shown to play
a key role in autonomic respiratory control (Ott et al., 2011). The
VRC is located in the ventrolateral medulla (VLM) and contains the
RTN, Btzinger complex (BC), pre-Btzinger complex (PBC), rostral
ventral respiratory group (rVRG), and caudal ventral respiratory
group (cVRG; Feldman and McCrimmon, 2008). The PBC contains
at least four kinds of inspiratory and expiratory neurons consisting of rhythm-generating microcircuits, and the BC transmits the
rhythms generated to lower neurons (Smith et al., 1991, 2007; Sun
et al., 1998; Koshiya and Smith, 1999). The rVRG and cVRG also
contain interneurons leading to the rostral medulla oblongata and
bulbospinal respiratory premotor neurons for the control of motor
neurons (Ellenberger and Feldman, 1990b; Ellenberger et al., 1990;
Kalia, 1981).
In addition to the VRC, noradrenergic A1 neurons and adrenergic C1 neurons are also involved in respiratory control in the
medulla oblongata. A1/C1 neurons are located ventral to the VRC,
and some A1/C1 neurons are intermingled with neurons in the BC
and rVRG/cVRG, respectively (Ellenberger et al., 1990). Previous
studies reported that A1/C1 neurons were activated by hypoxic
exposure because Fos-positive cells were observed in the A1/C1
neurons of rats exposed to hypoxia (Erickson and Millhorn, 1994;
Smith et al., 1995). These ndings indicated that A1/C1 neurons may
contribute to respiratory responses to changes in environmental
gas.
Although respiratory changes and the regulatory system in
the central circuit for respiratory regulation have not yet been
examined in detail under hypoxic and hypercapnic exposure, the
activities of the VLM have been suggested to be modulated by gas
exposure. In the present study, we examined respiratory responses
to hypoxia (10% O2 ), hypercapnia (10% CO2 ), and hypercapnic
hypoxia (10% O2 10% CO2 ) using plethysmography. The topographical expression pattern of Fos, a marker of neuronal activity, was
determined in the respiratory centers of rats exposed to these gases
for 2 h. We focused on the interrelationship between respiratory
responses and Fos expression in the VLM in order to elucidate the
neural mechanisms underlying hypoxia- and hypercapnia-evoked
respiratory responses.
2. Materials and methods
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Animal experimental protocols were approved by the Committee on the Use of Live Animals in Teaching and Research of Iwate
University (approval number: A201047).
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2.1. Head-out plethysmography
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2.1.1. Gas exposure experiments

Male Wistar rats (8 weeks old) were divided into four experimental groups (n = 4 in each group) were exposed to different
gases: 1) hypoxic group (10% O2 balanced N2 ), 2) hypercapnic group (20% O2 and 10% CO2 balanced N2 ), 3) hypercapnic
hypoxic group (10% O2 and 10% CO2 balanced N2 ), and 4) control group (20% O2 balanced N2 ). These gas exposure conditions
were decided by reference to previously studies (Wakai et al., 2010;
Ghannouchi et al., 2013; Takakura and Moreira, 2013; Lemes and
Zoccal, 2014; Damasceno et al., 2015). Each rat was anesthetized
with urethane (1.5 g/kg; intraperitoneal injection) and placed into
a head-out plethysmograph-chamber inside an acrylic chamber
(50 40 50 cm). Three holes, located at the top of a sidewall of
the acrylic chamber, were connected to three types of gas bombs
(O2 , CO2 , and N2 ). O2 and CO2 levels within the chamber were
monitored with a gas analyzer and each gas level was maintained
Fig. 1. Schematic illustration of the method for head-out plethysmograph.

A respiration waveform was obtained by detecting the inow and outow of air in
the plethysmograph-chamber using a ow head and spirometer pod connected to
PowerLab. When gasses owed from the inlet, the head of animal was exposed to
the gas. Gas concentration in the acrylic chamber was adjusted by N2 , CO2 and/or
O2 gasses that automatically regulated by solenoid valve (V) connected with O2 and
CO2 sensors.
automatically at a constant level. In the hypoxic experiment, O2

concentrations were maintained at 9.810.2% by N2 inux. In the
hypercapnic experiment, CO2 and O2 concentrations were maintained at 9.610.4% by CO2 inux and at 2020.5% by O2 inux,
respectively. In the hypercapnic hypoxic experiment, O2 and CO2
concentrations were maintained at 9.610.4% by N2 inux and at
9.610.4% by CO2 inux, respectively. In the control experiment, O2
concentrations were maintained at 2020.5% by O2 inux and CO2
concentrations were maintained at <1% by N2 inux. The temperature within the chamber was maintained at 25 C.
2.1.2. Measurement of respiration in rats exposed to each gas
Respiration measurements were collected in all rats for 30 min
before gas exposure. Gas exposure experiments and respiration
measurements were examined for 60 min. We measured changes
in the respiration of rats by detecting changes in the internal pressure of the plethysmograph-chamber using a respiratory ow head
(MLA 1L, ADInstruments, Sydney, Australia) and spirometer pod
(ML311, ADInstruments) connected to PowerLab (ADInstruments).
The experimental equipment for head-out plethysmograph is illustrated in Fig. 1. Respiration data were analyzed by LabChart 7
(ADInstruments). Flow volume was calibrated according to the
manufacturers protocol. In order to obtain a respiration waveform
for respiratory volume, we integrated a waveform of changes in
the internal pressure of the plethysmograph chamber. Respiratory
parameters obtained by plethysmography have high correlation
with the parameters obtained by pneumotachography in mice or
rats (Onodera et al., 1997; Nirogi et al., 2012; Hoymann, 2012).
Therefore, we considered the respiratory volume obtained in the
present study was comparable to tidal volume. We examined respiratory frequencies, tidal volumes, and respiratory minute volumes
for 1 min before and at ve time points after (0, 15, 30, 45 and 60 min
after exposure to each gas) from the waveforms of respiratory volume.
2.2. Statistical analysis
Statistical analyses were performed using the KruskalWallis
test with post hoc test (GamesHowel test) with p < 0.05 being
considered signicant.
2.3. Immunohistochemistry
2.3.1. Fos-immunohistochemistry
Male Wistar rats (8 weeks old) were divided into four experimental 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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facial nucleus and ventrolateral to the paragigantocellular reticular nucleus lateral part (PGRN). The mVLM is a part of the PGRN
and is located caudal to the facial nucleus, rostral to the LRN, and
ventral to the nucleus ambiguus (AMB). The cVLM is located caudal to the LRN and regions that enclose the LRN and AMB. Guyenet
and Wang (2001), Stornetta et al. (2009), and Wang et al. (2001)
reported that the rVLM includes the RTN, the mVLM includes the
BC and PBC, and the cVLM includes the rVRG and cVRG. The numbers of positive neurons were counted in all sections stained for
Fos-immunohistochemistry within the range of rVLM, mVLM or
cVLM. The cell counting was performed bilaterally on each section
under a light microscope. Statistical analyses were performed using
the KruskalWallis test with post hoc test (Games-Howel test) with
p < 0.05 being considered signicant.
Fig. 2. Location of the rVLM, mVLM, and cVLM.

This gure shows a sagittal section of medulla oblongata. In this study, we observed
the rVLM, mVLM, and cVLM. The rVLM is located ventral to the facial nucleus. The
mVLM is located caudal to the facial nucleus and ventral to the AMB. The mVLM
is located caudal to the mVLM and dorsal to the LRN. SO: Superior olive, VII: facial
nucleus, AMB: nucleus ambiguus, LRN: lateral reticular nucleus, rVLM: rostral ventrolateral medulla, mVLM: medial ventrolateral medulla, cVLM: caudal ventrolateral
medulla
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25 C in a cage (16 27 13 cm; free-feeding) inside the acrylic

chamber. After the exposure experiments, rats were anesthetized
with pentobarbital sodium (50 mg/kg; intraperitoneal injection)
and perfused transcardially with Ringers solution (200 mL) followed by Zambonis xative (4% paraformaldehyde, 0.5% picric acid
in 0.1 M phosphate buffer; pH 7.4). The brains were removed and
immersed in the same xative overnight at 4 C. They were then
rinsed (3 10 min) in phosphate buffered saline (PBS; pH 7.4),
soaked in 30% sucrose in PBS, and frozen. Serial transverse sections (50 m) were cut on the cryostat. The sections were collected
alternatively and divided two series. One series of sections was
processed for Nissl stain while the other was used for immunohistochemistry (avidin-biotin-peroxidase complex method; ABC
method). Nissl stain sections were used to identify medullary
nuclei.
Regarding enzyme immunohistochemistry, free-oating sections were rst rinsed in 0.5% Triton X-100 in PBS overnight at 4 C.
In order to block non-specic peroxidase activity, the sections were
incubated in PBS containing 1.5% H2 O2 for 1 h at room temperature
and then rinsed in PBS (3 10 min). The sections were then incubated for 72 h at 4 C with rabbit polyclonal antiserum against Fos
(Ab-5, 1:10,000, Oncogene Research Products, Cambridge, MA) and
non-immune donkey serum (1:50) to prevent non-specic binding sites. After being incubated, the sections were rinsed in PBS
(3 20 min) and incubated for 1 h in biotinylated anti-rabbit IgG
donkey serum (1:1000, Jackson ImmunoResearch Products, West
Grove, PA) and washed in PBS (3 20 min). The sections were
then incubated in an avidin-biotin-peroxidase complex (Vectastain
Elite ABC kit; Vector Laboratories, Burlingame, CA) for 2 h. After
washing with PBS (3 20 min), sections were incubated in 0.02%
3,3 -diaminobenzidine tetrahydrochloride in TrisHCl buffer solution (pH 7.4) in the presence of 0.0006% H2 O2 for 1530 min at
room temperature. Finally, after two washes in PBS, sections were
mounted on gelatin-coated slides, air-dried in an incubator at 37 C,
dehydrated in alcohol, cleared in xylene, and coverslipped.
For observation, the ventrolateral medulla was conveniently
divided into three parts, i.e., the rostral, medial, and caudal parts
(rVLM, mVLM, cVLM; Fig. 2). The rVLM is located ventral to the
2.3.2. Double immunouorescence for Fos and DBH

Some neurons belonging to noradrenergic A1 and adrenergic
C1 groups were previously shown to be intermingled with neurons of the mVLM and cVLM (Ellenberger et al., 1990). To clarify
whether Fos-positive neurons were catecholaminergic neurons or
non-catecholaminergic neurons, we performed double staining for
Fos and dopamine -hydroxylase (DBH), which is an indicator of
noradrenergic or adrenergic neurons. For indirect immunouorescence, serial transverse sections (20 m) from additional animals
of each group were cut on the cryostat, mounted on slides, and
air-dried for 12 h. Sections were rinsed in PBS (3 5 min) and
incubated for 24 h at 4 C with rabbit polyclonal antiserum against
Fos (Ab-5, 1:5000, Oncogene Research Products, Cambridge, MA),
mouse monoclonal antiserum against DBH (MAB308, Chemicon
International, 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 antirabbit IgG (1:200, Jackson ImmunoResearch Products, West Grove,
PA) and Cy3-conjugated donkey anti-mouse IgG (1:200, Jackson
ImmunoResearch Products, West Grove, PA), and then washed
in PBS (3 20 min). Sections were then coverslipped with Fluoromount (Diagnostic BioSystems, Pleasanton, CA). The sections
stained by double immunouorescence were observed under a
confocal laser microscope (C2, Nikon, Tokyo). All images were analyzed 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-out
plethysmography
The representative waveform of ow and volume derived from
plethysmography 30 min after gas exposure is shown in Fig. 3.
Table 1 shows tidal volumes, respiratory frequencies, and respiratory minute volumes at each time point in rats exposed to hypoxia,
hypercapnia, hypercapnic hypoxia, and control gas. When rats were
exposed to hypoxia, respiratory frequency was signicantly higher
at 15 min and 30 min, while respiratory minute volume was signicantly higher at 15 min than at 0 min. Tidal volumes and respiratory
minute volumes in rats exposed to hypercapnia and hypercapnic
hypoxia were signicantly higher at 15, 30, 45 and 60 min than at
0 min.
Respiratory minute volume, tidal volume, and respiratory frequency values at 0 min were set to 100%, and compared with each
value 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 hypoxia
than in those in the control group at 15, 30 and 45 min. Greater
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Fig. 3. Respiration waveform of changes in ow and volume 30 min after exposure to each gas.
In rats exposed to hypoxia, the amplitude of both ow and volume are similar between pre-exposure and 30 min after exposure, and respiration is increased (upper row). In
rats exposed to hypercapnia and hypercapnic hypoxia, the amplitude is increased while frequency remains unchanged (middle and lower rows).
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changes were induced in respiratory minute volume by hypercapnia or hypercapnic hypoxia than by hypoxia or the control gas
at all the time points examined. Tidal volume was enhanced to
approximately 270 and 250% in rats exposed to hypercapnia and
hypercapnic hypoxia, respectively, but was not enhanced in those
exposed to hypoxia (Fig. 4B). Tidal volume was signicantly higher
in rats exposed to hypercapnia or hypercapnic hypoxia than in rats
exposed to control gas in all the time points examined. On the
other hand, no signicant difference was observed in tidal volume

between the hypoxic and control groups. Respiratory frequency
was enhanced to 130150% in hypoxic rats and was signicantly
higher 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 not
enhanced in rats exposed to hypercapnia or hypercapnic hypoxia
(Fig. 4C).
Table 1
Changes of tidal volume, respiration frequency and respiratory minute volume during exposure of hypoxia, hypercapnia and hypercapnic hypoxia.
0 min
Tidal volume (mL)
Hypoxia
Hypercapnia
Hypercapnic hypoxia
Control
Respiratory frequency (breath/min)
Hypoxia
Hypercapnia
Hypercapnic hypoxia
Control
Respiratory minute volume (mL/min)
Hypoxia
Hypercapnia
Hypercapnic hypoxia
Control
15 min
0.37
0.40
0.52
0.45
0.031
0.047
0.035
0.038
125.8
131.5
120.8
114.5
46.6
51.8
62.5
51.1
30 min
0.35
1.03
1.22
0.44
0.031
0.166*
0.098*
0.058
17.5
9.3
9.1
23.4
183.0
138.5
139.3
108.0
3.9
6.6
3.6
10.5
63.0
142.0
169.3
47.2
45 min
60 min
0.34
1.11
1.31
0.44
0.03
0.10*
0.16*
0.09
0.35
1.12
1.32
0.41
0.03
0.11*
0.18*
0.08
0.34
1.10
1.31
0.41
0.02
0.09*
0.21*
0.07
20.0*
5.9
4.6
17.6
184.5
137.0
124.5
110.8
19.2*
7.8
12.6
18.5
167.3
137.3
122.5
115.0
16.8
5.4
16.1
28.8
167.8
140.1
123.0
119.5
26.2
10.7
22.7
10.5
8.1*
16.3*
8.0*
8.8
62.2
152.0
161.3
47.7
7.9
7.4*
4.4*
8.9
58.8
153.5
159.0
45.8
7.6
11.6*
6.2*
8.7
57.4
154.0
157.5
45.2
8.3
13.9*
5.8*
8.0
Hypoxia, 10% O2 ; Hypercapnia, 10% CO2 ; Hypercapnic hypoxia, 10% O2 10% CO2 .
Average S.D.
*
Signicant vs 0 min (pre-exposure term) at p < 0.05.
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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 hypercapnia
and hypercapnic hypoxia, respiratory minute volume and tidal volume are enhanced to 200% at all the exposure times examined, whereas respiratory frequency remains
unchanged. In rats exposed to hypoxia, respiratory minute volume and respiratory frequency are signicantly increased at 15, 30 and 45 min and tidal volume remains
unchanged. *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 control
groups, *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 are
expressed 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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Fig. 6. Fos immunoreactivity in the mVLM.

Fos-positive neurons are widely distributed in the PGRN of rats exposed to hypoxia (A), hypercapnia (B), and hypercapnic hypoxia (C). Fos-positive neurons were not observed
in control rats (D).
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3.2. Fos-positive neurons in the VLM

Few Fos-positive neurons were detected in the surface region
in hypoxia exposed and control rats, and the numbers of positive neurons were 9.7 4.0 and 7.0 6.4, respectively (Fig. 5A, D).
Numerous Fos-positive neurons were detected in the rVLM of rats
exposed to hypercapnia and hypercapnic hypoxia, and positive
neurons accounted for 317.8 25.6 and 363.2 25.6 per area,
respectively (Fig. 5B, C). Fos-positive neurons were also observed
in the RTN ventral to the facial nucleus including the ventral surface of the medulla. The numbers of Fos-positive neurons were
signicantly higher in rats exposed to hypercapnia and hypercapnic hypoxia than in rats exposed to hypoxia or the control gas
(Fig. 8A). No signicant differences were observed between the
Fig. 7. Fos immunoreactivity in the cVLM.

Fos-positive neurons are observed in the PGRN surrounded by the AMB and LRN in sections from rats exposed to hypoxia (A) and hypercapnic hypoxia (C). Few Fos-positive
neurons appear in sections from rats exposed to hypercapnia (B) and control rats (D).
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hypercapnic group and hypercapnic hypoxic group or between the

hypoxic group and control group.
Fos-positive neurons were detected in the mVLM of rats exposed
to hypoxia (207.0 32.1 per area), hypercapnia (297.7 73.5), and
hypercapnic hypoxia (296.2 64.7) (Fig. 6AC). Only a few Fospositive neurons were observed in the mVLM of the control group
(19.8 18.0) (Fig. 6D). In rats exposed to hypoxia, Fos-positive neurons were concentrated in the ventral part, while those in rats
exposed to hypercapnia or hypercapnic hypoxia broadly extended
throughout the mVLM. The mean numbers of Fos-positive neurons
in the mVLM were signicantly higher in the three experimental
groups than in the control group (Fig. 8B). The number of Fospositive neurons was signicantly lower in hypoxic rats than in
rats exposed to hypercapnia and hypercapnic hypoxia.
Many Fos-positive neurons were observed around the AMB
in the cVLM of rats exposed to hypoxia or hypercapnic hypoxia
(Fig. 7A, C). Few positive neurons were detected in the hypercapnic
or control group (Fig. 7B, D). As shown in Fig. 8C, the number of Fospositive neurons was signicantly higher in rats exposed to hypoxia
(364.8 69.5 per area) or hypercapnic hypoxia (187.5 81.2) than
in hypercapnic (55.3 36.4) or control rats (13.3 12.9). Furthermore, a signicant difference was noted between hypoxia and
hypercapnic hypoxia, but not between hypercapnia and the control.
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3.3. DBH immunoreactivity in Fos-positive neurons in the VLM
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Fos-positive neurons in the rVLM of rats exposed to hypercapnia

and hypercapnic hypoxia did not show immunoreactivity for DBH
(Fig. 9A). Some Fos-positive neurons in the mVLM of all groups
were also immunoreactive for DBH; however, Fos single positive
neurons were also observed (Fig. 9B). In the cVLM of rats exposed
to hypoxia or hypercapnic hypoxia, most Fos-positive neurons were
also positive for DBH (Fig. 9C, D). A few Fos-positive neurons did
not show immunoreactivity for DBH.
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4. Discussion
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4.1. Methodological considerations
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In the present study, measurement of respiration in the rat

exposed to each gas was performed under urethane anesthesia.
While urethane anesthesia is known its minimal effects on cardiovascular and respiratory systems and maintenance of spinal
reexes (Maggi and Meli, 1986; Hara and Hariis, 2002), animals under urethane anesthesia are used as a model of sleep
(Pagliardini et al., 2013). Respiration pattern of urethane anesthetized animals show similar to that of non-REM sleep animals,
i.e., decrease of minute ventilation and respiratory frequency is
decreased (Pagliardini et al., 2013). Thus, it may be the respiration
patterns are more or less different between urethane-anesthetized
and conscious animal. In the present study, respiratory responses to
hypoxia, hypercapnia or hypercapnic hypoxia were distinctly different. On the other hand, immunohistochemical analysis showed
experiment-specic distribution of Fos-immunoreactive nerve cell
bodies in the rat exposed to hypoxia, hypercapnia and hypercapnic
hypoxia in comparison to control. Thus, we consider that both physiological and immunohistochemical analyses would be represent
the effect of three different environmental gasses.
4.1.1. Localization of Fos-positive neurons in rVLM
Previous studies observed Fos-positive neurons in the RTN
of mice and cats exposed to hypercapnia (Niblock et al., 2012;
Teppema et al., 1994). In the present study, Fos-positive neurons
were observed in the rVLM including the RTN of rats exposed to
hypercapnia or hypercapnic hypoxia, but not in rats exposed to
hypoxia. CO2 /H+ sensitive neurons have been identied in the RTN
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 signicant higher with hypercapnia and hypercapnic hypoxia than with hypoxia and control gas. In the mVLM,
the number of Fos-positive cells is signicantly higher in the experimental groups
for gas exposure than in the control. In the cVLM, the number of Fos-positive cells
is signicantly higher in the hypoxic group than in the other groups, and is higher
in the hypercapnic hypoxia group than in the hypercapnia and control groups. 1:
p < 0.05 vs hypoxic group, 2: p < 0.05 vs hypercapnic group, 3: p < 0.05 vs hypercapnic
hypoxic 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 respiratory responses to hypercapnia (Akilesh et al., 1997). The present
results are in good agreement with these reports. On the other
hand, although the ring rate of RTN neurons was reported to be
increased by the carotid body with hypoxia (Takakura et al., 2006),
RTN was not activated in the present study. Thus, RTN neurons
may not play signicant roles in respiratory regulation for hypoxic
exposure.
RTN neurons were found to project to the mVLM, especially the BC and PBC, by the anterograde tracer biotinylated
dextran amine, and a large part of these projection neurons contained vesicular glutamate transporter 2 and galanin (Rosin et al.,
2006; Bochorishvili et al., 2012). Since the BC and PBC contain
rhythmogenic microcircuits for respiration (Smith et al., 2013),
excitatory neurotransmitters such as glutamate in the RTN may
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Fig. 9. Double immunouorescence for Fos and DBH in the VLM.

(A) The rVLM of rats exposed to hypercapnia. Fos-positive neurons (green) in the RTN are not immunoreactive for DBH. (B) The mVLM of rats exposed to hypercapnic hypoxia.
Double positive neurons (arrows) and neurons positive for Fos, but not for DBH are shown (arrowhead). (C, D) The cVLM of rats exposed to hypoxia. Double positive neurons
are concentrated. Some neurons that immunoreacted with Fos, but not with DBH are also shown (arrowhead). (F) is a higher magnication view of (E).
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affect respiratory rhythms to regulate respiratory minute volume

and tidal volume under hypercapnic conditions.
4.1.2. Localization of Fos-positive neurons in the mVLM
A close relationship has been reported between the activity of
the phrenic nerve and neurons in the mVLM or cVLM (Smith et al.,
2000). The PBC displays autonomic ring prior to inspiration, and
the BC contributes to the switch from inspiration to expiration
(Ezure et al., 2003; Shen et al., 2003; Smith et al., 2007). Furthermore, the mVLM contains several distinct respiratory neuron types
including excitatory and inhibitory neurons to make microcircuits
for rhythmogenesis (Schwarzacher et al., 1991; Shen et al., 2013;
Smith et al., 2013).
A previous study demonstrated that the administration of the
GABAB receptor agonist baclofen to the mVLM induced an increase
in the respiratory frequency of rabbits (Bongianni et al., 2010). The
respiratory frequency of rats exposed to hypoxia was increased in
the present study; therefore, Fos-positive neurons in the mVLM
may contain GABAergic inhibitory neurons. On the other hand, the
administration of glutamate to the PBC has been shown to induce
an increase in the respiratory minute volume of rats (Moraes et al.,
2011). In rats exposed to hypercapnia and hypercapnic hypoxia,
Fos-positive neurons in the mVLM may also contain excitatory glutamatergic neurons because respiratory minute volume and tidal
volume were elevated during gas exposure. The smaller number
of Fos-positive neurons in hypoxic rats suggests that excitatory
neurons are limited. Furthermore, the disruption of neurotransmission in the mVLM by the administration of colchicine was reported
to inhibit the increases induced in tidal volume, respiratory frequency, and respiratory minute volume by an exposure to hypoxia
or hypercapnia (Wu et al., 2005). Our results suggest that the mVLM
plays a key role in respiratory modulation by both hypoxia and
hypercapnia.
4.1.3. Localization of Fos-positive neurons in the cVLM
In the cVLM, excitatory premotor neurons exist between rhythmogenic microcircuits in the BC and PBC (Smith et al., 2013).
The rostral VRG and caudal VRG in the cVLM contain bulbospinal
premotor neurons, and the premotor neurons project to inspiratory motor neurons and expiratory motor neurons, respectively
(Smith et al., 2013). In addition to the premotor neurons in the
VRG, catecholaminergic A1/C1 neurons have been detected along
the VRG (review see Guyenet et al., 2013). In the present study,
most Fos-positive neurons in the cVLM may be A1/C1 neurons
because they showed DBH immunoreactivity. The presence of
Fos-positive neurons in rats exposed to hypoxia and hypercapnic hypoxia suggests that A1/C1 neurons were activated by the
hypoxic stimulation. The present results are consistent with previous ndings published by Smith et al. (1995), in which Fos
immunoreactivity was observed in A1/C1 neurons in the cVLM of
rats exposed to hypoxia. Furthermore, few Fos-positive neurons
were observed in rats exposed to hypercapnia, which indicates that
the activation of A1/C1 neurons may specic to hypoxia, not to
hypercapnia. A previous study reported that A1/C1 neurons exhibited Fos immunoreactivity following an electrical stimulus to the
carotid sinus nerve, which mimicked hypoxic exposure (Erickson
and Millhorn, 1994). Moreover, projections to the cVLM from the
NTS, which is the receiving input of the carotid sinus nerve, were
identied in tracer experiments (Aicher et al., 1995). Therefore,
Fos-positive A1/C1 neurons in the cVLM may mediate inputs from
the carotid body when animals are exposed to hypoxia, but not
to hypercapnia. On the other hand, retrograde tracer experiments
showed projections to the mVLM from the cVLM (Ellenberger and
Feldman, 1990a). Moreover, the administration of the adrenaline
2 receptor antagonist, yohimbine, to the mVLM decreased the ring rate of the phrenic nerve in a medullary preparation of mice in
which the pons and dorsal medulla were removed (Zanella et al.,
2006). Based on these ndings, the nerve endings of A1/C1 neurons
in the mVLM may release catecholamine to enhance respiratory
frequency.
In conclusion, we speculate that hypoxia and hypercapnia modulated rhythmogenic microcircuits in the mVLM via
A1/C1 neurons in the cVLM and the RTN in the rVLM,
respectively.
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Respiratory Physiology & Neurobiology xxx (2015) xxxxxx

Contents lists available at ScienceDirect

Respiratory Physiology & Neurobiology

Differences in respiratory changes and Fos expression in the

Jun Wakai a , Daichi Takamura b , Ryosuke Morinaga c , Nobuaki Nakamuta c ,

Respiration rhythm is known to be affected by hypoxic or

increased both respiratory frequency and tidal volume. On the

J. Wakai et al. / Respiratory Physiology & Neurobiology xxx (2015) xxxxxx

2.1. Head-out plethysmography

2.1.1. Gas exposure experiments

Fig. 1. Schematic illustration of the method for head-out plethysmograph.

automatically at a constant level. In the hypoxic experiment, O2

Fig. 2. Location of the rVLM, mVLM, and cVLM.

25 C in a cage (16 27 13 cm; free-feeding) inside the acrylic

2.3.2. Double immunouorescence for Fos and DBH

J. Wakai et al. / Respiratory Physiology & Neurobiology xxx (2015) xxxxxx

other hand, no signicant difference was observed in tidal volume

Fig. 5. Fos immunoreactivity in the rVLM.

Fig. 6. Fos immunoreactivity in the mVLM.

3.2. Fos-positive neurons in the VLM

Fig. 7. Fos immunoreactivity in the cVLM.

hypercapnic group and hypercapnic hypoxic group or between the

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

Fos-positive neurons in the rVLM of rats exposed to hypercapnia

4.1. Methodological considerations

In the present study, measurement of respiration in the rat

using patch clamp techniques (Lazarenko et al., 2009). Moreover,

Fig. 9. Double immunouorescence for Fos and DBH in the VLM.

affect respiratory rhythms to regulate respiratory minute volume

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