JOURNAL OF NEUROCHEMISTRY | 2011 | 117 | 961–972
CRN2M, UMR 6231, Centre National de la Recherche Scientifique, Université Paul Cézanne, Université de la Méditerranée,
Marseille, France
Abstract
Ozone (O3), a major component of air pollution, has consid-
erable impact on public health. Besides the well-described
respiratory tract inflammation and dysfunctions, there is
accumulating evidence indicating that O3 exposure affects
brain functions. However, the mechanisms through which O3
exerts toxic effects on the brain remain poorly understood.
This work aimed at precisely characterizing CNS neuronal
activation after O3 inhalation using Fos staining in adult rat.
We showed that, together with lung inflammation, O3 expo-
sure caused a sustained time- and dose-dependent neuronal
activation in the dorsolateral regions of the nucleus tractus
solitarius overlapping terminal fields of lung afferents running
in vagus nerves. Furthermore, we highlighted neuronal
Tropospheric ozone (O3), a major environmental component
of photochemical urban air pollution formed from atmo-
spheric pollutants, is detrimental to health because of its
oxidant and toxic properties (reviewed in Mudway and Kelly
2000). Depending on geographical and seasonal factors, O3
baseline concentration naturally occurring in the troposphere
varies between 0.02 and 0.04 ppm (part per million), which
is only three or five times below deleterious concentrations.
Exposure to this toxic gas is more and more frequent within
and around urban areas, and contributes significantly to
increase morbidity in human populations (Gryparis et al.
2004; Chang et al. 2010; Cooper et al. 2010).
Although most reports on the effects of O3 have focused
on respiratory tract inflammation and dysfunctions (reviewed
in Mudway and Kelly 2000), there is accumulating evidence
that supra-ambient levels of O3 could affect the CNS,
resulting not only in neuronal changes in the brainstem
(Chen et al. 2003) but also in memory impairment (Avila-
Costa et al. 1999), disruption of sleep patterns (Paz and
Bazan-Perkins 1992), reduced cognitive performance (Tep-
per and Weiss 1986; Chen and Schwartz 2009), alteration of
social behavior (Musi et al. 1994), and motor activity deficits
(Dorado-Martı́nez et al. 2001). However, the mechanisms
through which O3 exerts toxic effects on the CNS remain
 2011 The Authors
Journal of Neurochemistry  2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 961–972
doi: 10.1111/j.1471-4159.2011.07267.
activation in interconnected central structures such as the
caudal ventrolateral medulla, the parabrachial nucleus, the
central nucleus of the amygdala, the bed nucleus of the stria
terminalis and the paraventricular hypothalamic nucleus. In
contrast, we did not detect any neuronal activation in the
thoracic spinal cord where lung afferents running in spinal
nerves terminate. Overall, our results demonstrate that O3
challenge evokes a lung inflammation that induces the acti-
vation of nucleus tractus solitarius neurons through the vagus
nerves and promotes neuronal activation in stress-responsive
regions of the CNS.
Keywords: brain mapping, inflammation, NTS, ozone,
stress.
J. Neurochem. (2011) 117, 961–972.
poorly understood; and the possible involvement of a neural
pathway linking respiratory tract inflammation to CNS
dysfunctions has never been examined. The current study
has been designed to establish the pattern of central neuronal
activation induced by O3 inhalation. In this context, c-Fos
and Fos-B immunoreactive (Fos-ir) staining was used to
reveal activated neurons (reviewed in Kovács 1998).
Received December 15, 2010; revised manuscript received February 28,
2011; accepted March 31, 2011.
Address correspondence and reprint requests to Dr Florian Gackière,
CRN2M, UMR 6231, Faculté de Médecine Nord, CS80011, Bd Pierre
Dramard, 13344, Marseille Cedex 15, France.
E-mail: florian.gackiere@univmed.fr
1
The present address of Agnès Baude is the INMED, INSERM U901,
Université de la Méditerranée, Parc Scientifique de Luminy, 13273
Marseille, France.
Abbreviations used: AP, area postrema; BAL, bronchoalveolar lavage;
BST, bed nucleus of the stria terminalis; CeA, central nucleus of the
amygdala; CVLM, caudal ventrolateral medulla; DR, dorsal raphe
nucleus; IL, interleukin; LC, locus cœruleus; NTS, nucleus tractus
solitarius; O3, ozone; PB, phosphate buffer; PBN, parabrachial nucleus;
PBS, phosphate buffer saline; PBST, PBS containing 0.3% Triton
X-100; PVN, paraventricular hypothalamic nucleus; TH, tyrosine
hydroxylase.
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962 | F. Gackière et al.

The nucleus tractus solitarius (NTS), a structure located in
the dorsal brainstem, is known to be critical for homeostasis
and autonomic regulation. We especially explored it as it is
the major gateway through which visceral primary afferent
information (including that arising from the respiratory tract)
enters the brain, is integrated and ultimately relayed through
other brain structures to generate the respiratory reflexes
(Blessing 1997). Because the primary afferent innervation of
the respiratory tract is double (Springall et al. 1987; Plato
et al. 2006), being conveyed toward the NTS via the vagus
nerves and toward the spinal cord via the thoracic spinal
nerves, the thoracic spinal cord was also examined.
Materials and methods
Animals and ozone exposure
Experiments were performed on 5- to 6-week-old male adult Wistar
rats weighing between 150 and 220 g. All procedures were in
agreement with the European Communities Council directive (2010/
63/EC).
Rats were enclosed in their own cage in a transparent Plexiglas
chamber just before the exposure and were conscious during the
treatment. O3 was generated by passing air through a generator
regulated by a variable-voltage supply and delivered to the chamber.
O3 concentration was continuously monitored with an ultraviolet
analyzer (O3 41M, Environnement SA, Poissy, France). O3-exposed
animals inhaled 0.5 or 2 ppm ± 10% of O3 during 1.5–120 h (see
Table 1). Sham rats were subjected to the same protocol with the
same flow rate of ambient air, while control rats were not exposed.
The number of rats used for each type of experiment is summarized
in Table 1. All rats were deeply anesthetized by an intramuscular
injection of a mixture of ketamine (Imalgène 1000, 50 mg/kg;
Centravet, Lapalisse, France) and xylazine (Rompun 2%, 15 mg/kg;
Centravet) immediately after exposure. Different groups of rats were
used to measure lung inflammation, evaluate c-Fos and Fos-B
expression in the NTS, and investigate c-Fos expression in other
CNS regions.
The O3 concentrations employed were the ones usually used for
rats conditioning (between 0.5 ppm for intermittent long-term
exposures and 2–3 ppm for acute short-term exposures). Although
these doses are higher than those present in air quality standards (up
to 0.3 ppm for peak exposure limit in workrooms; reviewed in
Sandström 1995), this choice was justified when considering that O3
toxicity in resting rats is known to underestimate effects in
exercising humans (Hatch et al. 1994). Moreover, our primary goal
was not to mimic air pollution peaks, but rather to provide
fundamental data to describe the neural pathways activated by O3.
Measurements of pulmonary inflammation
The lung inflammatory response was determined using bronchoal-
veolar lavage (BAL) fluid analysis. The chest was opened to remove
the trachea and the lungs. A polyethylene tube connected to a
syringe was inserted into the trachea in order to inject 10 mL/kg
saline that was gently aspirated after lung lavage. The total number
of BAL leukocytes was counted manually under an optical
microscope using a Malassez slide.
Histological study
Tissue preparation
Rats were perfused through the ascending aorta with 200–300 mL
of 4% paraformaldehyde in phosphate buffer (PB, 0.1 M; pH 7.4).
Brain and spinal cord were removed and washed in PB. Medulla,
whole brain and thoracic spinal cord (T1–T6) coronal sections (40-
lm thickness) were obtained on a vibratome (VT-1000S, Leica,
Heidelberg, Germany), and collected serially in 24-well plates
containing PB added with 0.1% sodium azide.
Immunoperoxidase staining
CNS neuronal activation was investigated using the immediate-early
gene products, c-Fos and Fos-B, as immunohistochemical nuclear
markers. When acute challenges rapidly activate c-Fos expression,
Fos-B protein appears to accumulate on a longer time-scale
(reviewed in Kovács 1998). Immunostaining was performed on
free-floating sections incubated first for 30 min in PB containing
0.5% H2O2 to quench endogenous peroxidase and transferred for
1 h (20–23C) to 5% normal donkey serum (Jackson Immunore-
search Laboratories Inc., West Grove, PA, USA) in phosphate buffer
saline (PBS, PB containing 0.9% NaCl). Sections were then
incubated overnight at 4C with either goat anti-c-Fos (1/5000) or

Table 1 Number of animals used in each

O3 exposure 0h 1.5 h 3h 4.5 h 6h 15 h 24 h 72 h 120 h
experiment
BAL analysis
0.5 ppm 6 7 8 10 9
2 ppm 8 9 8 8 7 7
sham 9 7 5 3 3 6 4
Fos in NTS
0.5 ppm 5 3 4 3 4
2 ppm 2 5 4 4 4 3
sham 8 3 2 1 2
Mapping
2 ppm 1 4 1
sham 3 1 3 1

The table summarizes the different conditions implemented for O3 conditioning and the numbers of
animals used in each case to analyze BAL fluid (lines 1, 2, 3), c-Fos and Fos-B expression in the
NTS (lines 4, 5, 6), c-Fos expression in spinal cord and other CNS regions (lines 7, 8).

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Fos CNS mapping following ozone inhalation | 963

goat anti-Fos-B (1/3000) polyclonal antibodies [c-Fos (4) sc-52-G All results were given as mean ± SEM, n being the number of
or Fos-B (102) sc-48-G, respectively; Santa Cruz Biotechnology, rats per group.
Santa Cruz, CA, USA] diluted in PBS containing 0.3% Triton X-
100 (PBST). They were washed in PBS and successively incubated
for 2 h (20–23C) in anti-goat biotinylated secondary antibody Results
(705-065-147, Jackson Immunoresearch Laboratories; 1/400) in
PBST and in the avidin-biotin complex (ABC kit, Vector Labora- Assessing lung inflammation
tories, Burlingame, CA, USA; 1/200) in PB. Peroxidase activity was Within the first hours of O3 exposure, the number of
revealed using diamino-3,3¢-benzidine (0.25 mg/mL), nickel ammo- leukocytes recovered by BAL increased in a dose-dependent
nium sulphate (2 mM) intensification and H2O2 (0.01%) in PB. Sets manner before reaching a plateau after 24 h (68 ± 8 · 104
of sections from control, sham and experimental rats as well as and 119 ± 26 · 104 cells after 72 h of exposure to 0.5 and
sections incubated in the absence of primary antibody (to evaluate 2 ppm O3, n = 10 and 7, respectively) (Fig. 1). In contrast,
non-specific staining) were reacted in parallel. Finally, sections were
leukocyte numbers were similar in air-exposed sham and
rinsed, mounted on superfrost slides, air-dried, dehydrated in graded
control rats, never exceeding 26 · 104 cells. In agreement
alcohols, cleared in xylene and coverslipped with Di-N-butyle
phthalate in xylene (BDH Laboratory Supplies, Poole, UK).
with other studies (Pino et al. 1992; Bhalla and Gupta 2000;
van Bree et al. 2001), we found that O3 inhalation promoted
Immunofluorescence a persistent dose- and time-dependent lung inflammation in
In order to identify whether NTS catecholaminergic neurons were adult rats.
activated after O3 inhalation, we performed co-immunolabeling of
tyrosine hydroxylase (TH) and c-Fos. The protocol was as described Fos expression in NTS
above except that floating sections were sequentially co-incubated In the rats exposed 6 h to 2 ppm O3, the NTS exhibited
overnight at 4C with anti-c-Fos (see above; 1/1000) and monoclonal numerous unevenly distributed c-Fos-ir neurons. Most
anti-TH (mouse; MAB5280, Chemicon, Millipore, Billerica, MA,
labeled cells were detected in the dorsal part of the nucleus
USA; 1/5000) antibodies in PBST, washed before co-incubated in the
at the caudal level, as well as in the dorsomedial and
dark for 2 h (20–23C) with appropriate secondary antibodies
conjugated with either Alexa Fluor 488 (Al-488-donkey-anti-goat; dorsolateral parts at the subpostremal level and in the
Molecular ProbesTM, Invitrogen, Carlsbad, CA, USA; 1/500) or Cy3 dorsomedial and medial parts at the intermediate level
(Cy3-donkey-anti-mouse; Jackson Immunoresearch Laboratories; 1/ (Fig. 2d–f). In contrast, we did not observe any positive cells
200) in PBST. Finally, sections were rinsed, mounted on slides and at the rostral level of the nucleus (not shown). c-Fos-ir
coverslipped using glycerol/PB 0.2 M (vol/vol). neurons were also absent from the area postrema (AP) and
were very few in the dorsal motor nucleus of the vagus nerve.
Image acquisition and analysis Consistent with previous studies (Sagar et al. 1995; Teppema
Images were obtained with an inverted microscope (Zeiss AxioOb- et al. 1997; Yokoyama and Sasaki 1999), c-Fos expression
server Z1, Le Pecq, France) coupled to a camera (Zeiss AxioCam was virtually absent in the NTS of sham (Fig. 2a–c) and
Mrm) using a 10· objective (NA 0.25). Light transmitted images
control (not shown) animals, indicating that neuronal activa-
were acquired using a halogen lamp. Immunofluorescence images
tion observed in O3-exposed rats was specifically induced by
were acquired using a mercury arc lamp associated with Alexa-488
(470/40, 525/50) and Cy3 (545/25, 605/70) filter cubes. Acquisition O3 inhalation. In the same way, Fos-B-ir neurons increased
was performed with Axiovision software. Slightly overlapping in number in the NTS after O3 inhalation (Fig. 3d–f), when
images were taken bilaterally from each section to survey the lateral
extent of all structures of interest. Image editing was performed
using Adobe Photoshop (Adobe Systems, Paris, France) whereby
only brightness and contrast were adjusted. The NTS was sub-
divided into caudal, subpostremal, intermediate and rostral levels as
previously described (Barraco et al. 1992). Labeled brain regions
were identified according to the maps described in the stereotaxic
atlas by Paxinos and Watson (1998).
Peroxidase bright-field images were used to manually blind count
c-Fos or Fos-B positive nuclei in the NTS. Numbers given in the text
were relative and corresponded to the numbers of stained nuclei
counted in the whole NTS from a 40-lm thick slice at a given
rostrocaudal level. c-Fos mapping was performed on every fourth
section from the whole brain, separated by 160 lm. Semi-quantita-
tive counting of the number of TH-ir positive neurons was performed
manually; only neurons whose nuclei were visible were counted. Co- Fig. 1 Number of leukocytes in bronchoalveolar lavage (BAL) fluid in
labeling of TH and c-Fos proteins was analyzed in a total of five to rats exposed to 0.5 ppm or 2 ppm O3 for different durations, compared
seven caudal/subpostremal NTS sections per rat. c-Fos-ir neurons with sham rats. Control values are reported at 0 h with sham symbol.
profiles were scored and tested for TH, and inversely. The number of animals used in each condition is detailed in Table 1.

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Journal of Neurochemistry  2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 961–972

964 | F. Gackière et al.
(a)
(a′)
(b)
(c)
Fig. 2 Representative images of c-Fos immunoperoxidase labeling
within the NTS in sham rats (a–c) and in rats exposed 6 h to O3 (d–f).
a¢ and d¢ are higher magnifications of the areas delineated in (a) and
(d). Coronal sections of three caudorostral NTS levels are illustrated
[a-a¢ and d-d¢: caudal, (b) and (e): subpostremal, (c) and (f): inter-
mediate]. While virtually no c-Fos positive neurons can be seen in
Fos-B expression remained weak in the NTS of control (not
shown) and sham (Fig. 3a–c) rats. Fos-B distribution pattern
was similar to that described for c-Fos.
Fos-ir neurons were counted at each rostrocaudal level and
their number was plotted as a function of O3 exposure
duration (Fig. 4a and b). As shown in Figs 2 and 3, the
number of stained neurons was higher in the caudal and
subpostremal sections than in the intermediate ones. Analysis
of the effect of the O3-exposure duration showed that the
total number of c-Fos-ir neurons raised abruptly to 185 ± 31
(n = 5) after 3 h of exposure to 2 ppm O3, peaked up to
188 ± 37 (n = 4) after 6 h and declined thereafter to reach
51 ± 37 (n = 3) after 72 h (Fig. 4c). In contrast, the number
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(d)
(d′)
(e)
(f)
sham condition at each level (a–c), numerous c-Fos-ir neurons are
present in O3-exposed rats (d–f). Note that specific c-Fos-ir staining
induced by O3 exposure is circumscribed to the NTS. 4V, fourth
ventricle; AP, area postrema; CC, central canal; DMX, dorsal motor
nucleus of the vagus nerve; NTS, nucleus tractus solitarius; ts, tractus
solitarius. Scale bars = 100 lm.
of Fos-B-ir neurons reached a plateau for O3 exposures
longer than 15 h and persisted during the full time of
exposure (Fig. 4b). The total number increased from 49 ± 12
(n = 8) and 61 ± 47 (n = 8) in sham and control animals,
respectively, to 165 ± 37 (n = 5) and 265 ± 57 (n = 4) after
3 and 15 h of exposure to 2 ppm O3 (Fig. 4d).
c-Fos and Fos-B expression after an O3 challenge was
also dose-dependent. We did observe that a weaker dose of
O3, namely 0.5 ppm, was sufficient to promote an increase
in the number of Fos-ir neurons (Fig. 4c and d). Neuronal
activation induced by 0.5 ppm O3 was weaker but the
pattern was similar to that observed in response to a 2-ppm
O3 challenge, suggesting that the pathways involved were

(a)
(a′)
(b)
(c)
Fig. 3 Representative images of Fos-B immunoperoxidase labeling
within the NTS in sham rats (a–c) and in rats exposed 15 h to O3 (d–f).
a¢ and d¢ are higher magnifications of the areas delineated in (a) and
(d). Coronal sections of three caudorostral NTS levels are illustrated [a-
a¢ and d-d¢: caudal, (b) and (e): subpostremal, (c) and (f): intermediate].
Note that a few Fos-B positive neurons are observed in NTS sham
the same. In the following experiments, taking also into
account that c-Fos expression peaked around 6 h (Fig. 4c)
and was negligible in sham and control rat brain whereas
there was a weak basal Fos-B expression (Figs 2–4), we
analyzed c-Fos immunolabeling after 6 h of exposure to
2 ppm O3.
c-Fos expression in spinal cord
We did not detect any c-Fos-ir neurons along the T1–T6
thoracic spinal cord coronal sections in both sham rats
(Fig. 5a–c) and rats exposed during 6 h to 2 ppm O3
(Fig. 5d–f), indicating that the thoracic spinal cord pathway
was not involved in the transmission of the noxious O3
stimulus.
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Fos CNS mapping following ozone inhalation | 965
(d)
(d′)
(e)
(f)
condition at each level (a–c) and that O3 exposure promotes a large
increase in the number of NTS labeled neurons (d–f). Fos-B-ir staining
is dense in the NTS after O3 exposure while specific staining is lacking
in surrounding areas. 4V, fourth ventricle; AP, area postrema; CC,
central canal; DMX, dorsal motor nucleus of the vagus nerve; NTS,
nucleus tractus solitarius; ts, tractus solitarius. Scale bars = 100 lm.
c-Fos expression in catecholaminergic neurons
Co-immunolabeling for tyrosine hydroxylase (TH) and c-Fos
was used to determine whether the NTS catecholaminergic
neurons, known to project to higher brain regions, contrib-
uted to neuronal activation after the O3 challenge (Fig. 6). As
expected from the distribution of catecholaminergic neurons
in the brainstem (reviewed in Sawchenko and Swanson
1982), TH immunoreactive (TH-ir) neurons were mostly
found in the medial and ventral parts of the caudal and
subpostremal NTS levels (Fig. 6b). Although their distribu-
tion only partially overlapped that of NTS c-Fos-ir neurons,
some c-Fos positive neurons also exhibited TH co-labeling.
Most interestingly, the double-labeled cells represented
not less than 19 ± 4% of TH-ir positive cells (counting

966 | F. Gackière et al.
Fig. 4 Numbers of c-Fos-ir (a) and Fos-B-ir (b) neurons at the inter-
mediate, subpostremal and caudal NTS levels plotted as a function of
2-ppm O3 exposure duration. Each value corresponds to the mean of
measurements (n values are given in Table 1). The total numbers of
immunoreactive neurons, corresponding to the sum of c-Fos-ir (c) and
Fos-B-ir (d) neurons counted at the three anatomical NTS levels
performed on four animals) (Fig. 6a–d), indicating that a
substantial part of NTS catecholaminergic neurons was
activated by O3 inhalation.
c-Fos expression in other CNS regions
Results of the mapping of c-Fos-ir neurons in the whole
brain highlighted a set of central structures that presented
substantial numbers of activated neurons after exposition to
2 ppm O3 during 6 h (Fig. 7). In contrast, virtually no
positive neurons were found in sham or control rats (not
shown). In the brainstem, we observed c-Fos expression in
the caudal ventrolateral medulla (CVLM; Fig. 7a) but not in
the rostral ventrolateral medulla (not shown). In the pons, a
large c-Fos-ir staining was detected in the parabrachial
nucleus (PBN), namely in its external lateral part, whereas
nearly no expression was found in the medial PBN (Fig. 7b).
Moderate numbers of c-Fos-ir neurons were also present
within the Kölliker-Fuse nucleus (Fig. 7b) and few were
detected in the locus cœruleus (LC; Fig. 7c). In the midbrain,
c-Fos expression was found in the caudal and dorsal parts of
the dorsal raphe nucleus (DR; Fig. 7d). In the forebrain, a
strong c-Fos-ir staining was observed in the paraventricular
hypothalamic nucleus (PVN) where the labeled cells were
numerous in the parvocellular part but sparse in the
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(intermediate, subpostremal and caudal) from a same rat, are plotted
as a function of exposure durations. Each value corresponds to the
mean of measurements (n values are given in Table 1). Data reported
at 0 h corresponds to the average of all the values obtained from sham
and control.
magnocellular part (Fig. 7e). We also detected aggregated
c-Fos-ir neurons in both the central nucleus of the amygdala
(CeA; Fig. 7f) and the paraventricular thalamic nucleus
(Fig. 7g), as well as in the lateral part of the bed nucleus of
the stria terminalis (BST; Fig. 7h).
Discussion
The NTS as the primary relay structure of ozone effects
Air pollution impacts the CNS through diverse pathways
(reviewed in Block and Calderón-Garcidueñas 2009). The
link between O3-induced lung inflammation and brain
dysfunctions remained elusive. Our results revealed a
persistent time- and dose-dependent neuronal activation in
the NTS after O3 inhalation, supporting the involvement of
the vagus nerves and of the NTS in the processing of
information coming from the lungs. The number of activated
neurons after O3 exposure was more important in the dorsal
parts of the caudal and subpostremal NTS levels. Their
distribution overlapped extensively lung afferent terminal
fields arising from bronchopulmonary C fibers and inputs
from RAR (rapidly adapting stretch receptors) (reviewed in
Kubin et al. 2006). This pattern of Fos expression was
partially distinct from the patterns described in response to

(a)
(d)
Fig. 5 Representative images from the
thoracic spinal cord showing no c-Fos-ir
labeling either in the sham rats (a–c) or in
the rats exposed 6 h to O3 (d–f). The
coronal sections illustrated are from T1 (a
and d), T3/T4 (b and e) and T6/T7 (c and f)
levels of the spinal cord. Scale bars =
100 lm.
hypoxia or hypercapnia that show activation in the medial
and ventrolateral parts of the NTS (Teppema et al. 1997;
Kline et al. 2010). Altogether these results argue for a
specific activation of pulmonary afferent fibers by O3.
Sensory innervation of the lung is described to arise not
only from the vagus nerves projecting to the NTS, but also
from the spinal nerves entering the thoracic spinal cord
(Springall et al. 1987; Plato et al. 2006). Whatever the
conditions, we did not detect any neuronal activation along
the T1–T6 levels of the spinal cord. Overall, our results
showed that the signal induced by O3 exposure was conveyed
by the vagal primary afferents but not by the thoracic spinal
nerves. In accordance, previous studies established that O3
exposure stimulates RAR and C fibers (Coleridge et al. 1993;
Joad et al. 1998). In addition, Taylor-Clark and Undem
(2010) demonstrated that O3 directly activates Transient
Receptor Potential Ankyrin 1 in a subset of airway C fibers.
Together with neural pathways, humoral pathways
transduce signals from the periphery to the brain through
blood-borne agents, which reach the CNS through circum-
ventricular organs such as the AP. Molecules derived from
O3 and lung tissue interactions have been proposed to
mediate non-pulmonary O3 effects (reviewed in Block and
Calderón-Garcidueñas 2009). One hypothesis is that reactive
oxygen species formed secondarily to O3 exposure could
exert harmful CNS effects. Alternatively, various studies
 2011 The Authors
Journal of Neurochemistry  2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 961–972
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Fos CNS mapping following ozone inhalation | 967
(b) (c)
(e) (f)
point out that the O3 extra pulmonary effects may be
mediated by cytokines released from activated lung macro-
phages. Indeed, in rats, O3 challenge induces an increase in
interleukin (IL)-1b and tumor necrosis factor-a transcripts in
BAL fluid (Ishii et al. 1997), an activation of IL-1a, IL-6 and
IL-10 in lung tissue (Bhalla et al. 2002) and elevated levels
of tumor necrosis factor-a in brainstem (Araneda et al.
2008). However, in the current work, we did not detect any
significant neuronal activation in the AP, and the NTS did not
appear to be entirely activated but rather displayed a specific
pattern of Fos immuno-staining consistent with lung affer-
ents activation (Blessing 1997). These observations are not in
favor of the participation of blood-borne agents in the CNS
neuronal activation in response to O3-induced local lung
inflammation. They are in accordance with human studies
showing no increase in the level of systemic inflammatory
markers (circulating IL-6 and IL-8) after O3 exposure at low
concentrations known to induce lung inflammation (Hermans
et al. 2005; Urch et al. 2010). They are also in line with data
indicating that intrajejunal lipopolysaccharides injection,
which produces local visceral inflammation, does not elicit
activation of the AP (Gakis et al. 2009).
Functional circuits implicated in ozone effects
The mapping of CNS activated area strikingly showed that
O3 challenge promoted a consistent and reproducible pattern
 14714159, 2011, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.2011.07267.x by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [04/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
968 | F. Gackière et al.

(a) (a′)

(b) (b′)

(c) (c′)

Fig. 6 (a–c) Representative fluorescence

microscopy images of c-Fos (green; signal
in the nucleus, a and c) and tyrosine
hydroxylase (TH; red; signal in the cyto-
plasm, b and c) co-labeling within the NTS
(d)
of rats exposed 6 h to 2 ppm O3. a¢, b¢ and
c¢ are higher magnifications of the areas
delineated in (a), (b) and (c). On the merged
images (c, c¢) neurons expressing c-Fos
only (arrows), TH only (arrowheads) or both
c-Fos and TH (double arrowheads) are
shown. (d) Number of NTS TH-ir neurons
counted in each of the four rats analyzed. In
each case, the percentage of NTS TH-ir
neurons exhibiting c-Fos co-labeling
(TH + c-Fos) is indicated. AP, area
postrema; CC, central canal; DMX, dorsal
motor nucleus of the vagus nerve; NTS,
nucleus tractus solitarius; ts, tractus soli-
tarius. Scale bars = 100 lm.

of neuronal activation in anatomically interconnected brain- referred as ‘processive’ or ‘systemic’, and promoting differ-
stem and forebrain regions that are known to be implicated in ent patterns of activation, are usually distinguished (Yokoy-
the stress response. At least two major categories of stress ama and Sasaki 1999; Herman et al. 2003; Ulrich-Lai and

 2011 The Authors

Journal of Neurochemistry  2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 961–972
 14714159, 2011, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.2011.07267.x by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [04/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Fos CNS mapping following ozone inhalation | 969

(a) (b)

(c) (d)

(e) (f)
Fig. 7 Representative images showing c-
Fos immunoperoxidase labeling in various
brain regions after 6 h of exposure to 2 ppm
O3. Scale bars = 100 lm. (a) CVLM, caudal
ventrolateral medulla; lrn, lateral reticular
nucleus; nra, retroambiguus nucleus. (b)
PBNl, parabrachial nucleus, lateral part;
PBNel, PBN external lateral part; PBNm,
PBN medial part; scp, superior cerebellar
pedoncule; KF, Kölliker-Fuse nucleus. (c)
LC, locus cœruleus; 4V, fourth ventricle;
Me5, mesencephalic nucleus of the tri-
geminal nerve. (d) DRc, dorsal raphe nu-
cleus, caudal part; pag, periaqueductal
gray; aq, cerebral aqueduct. (e) PVN, pa- (g) (h)
raventricular hypothalamic nucleus; pv,
PVN parvocellular part; mg, PVN magno-
cellular part; mpo, medial pre-optic nucleus;
3V, third ventricle. (f) CeA, central nucleus
of the amygdala; ic, internal capsule; bla,
basolateral nucleus of the amygdala; cpu,
caudate putamen. (g) PVT, paraventricular
thalamic nucleus; 3V, third ventricle; sm,
stria medullaris of thalamus. (h) BSTl, bed
nucleus of the stria terminalis, lateral part;
cpu, caudate putamen; ac, anterior com-
missure; ls, lateral septum; V, lateral ven-
tricle.

 2011 The Authors

Journal of Neurochemistry  2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 961–972

970 | F. Gackière et al.
Herman 2009). The ‘processive’ stress (also called emo-
tional, exteroceptive or psychological) relies on somatosen-
sory/nociceptive inputs and involves an affective emotional
component often requiring a degree of interpretation by
cortical structures. It may be experimentally induced by
treatments such as restraint, fear conditioning, forced swim
or exposure to a novel environment, which do appear to be
channeled through limbic forebrain regions that ultimately
control PVN activity. By contrast, the ‘systemic’ stress (also
called physical, interoceptive or homeostatic), such as the
stress promoted by infection, cytokines or intraperitoneal
lipopolysaccharide administration, targets homeostatic
parameters and does not require cognitive processing. It
involves a reflexive response relayed directly to the PVN via
brainstem catecholaminergic projections.
In the present work, we showed that a subset of NTS
catecholaminergic neurons (almost 20%) was activated by O3
challenge. These neurons are ideally located to relay O3-
induced NTS neuronal activation to multiple other brain
regions, since they are known to project to higher central
autonomic nuclei such as the CVLM, PBN, PVN, BST and
CeA (Gaykema et al. 2007; Hermes et al. 2006; and
reviewed in Hollis et al. 2004). The patterns of neuronal
activation we observed in these regions are consistent with
this view. We did find a significant activation in the CVLM,
which is a NTS direct projection site and a part of autonomic
reflexes pathways (Hermes et al. 2006). The external lateral
PBN, another NTS direct projection site, was also strongly
activated whereas the medial PBN was not. The lateral PBN
constitutes an important relay to transfer ascending viscero-
sensory information from the NTS visceral part to higher
order centers (hypothalamus and forebrain regions) involved
in autonomic regulation and self-awareness (Hermes et al.
2006; Geerling et al. 2010). Few neurons were activated in
the Kölliker-Fuse nucleus, which contains various respira-
tion-related neurons (Ezure and Tanaka 2006). In the
hypothalamus, the major site of O3-induced neuronal
activation was the PVN parvocellular part, known to form
an essential link between limbic functions and autonomic
functions (including respiratory activities), and to be com-
posed of neurons forming the common final pathway of the
stress response (Geerling et al. 2010). The LC, DR and
paraventricular thalamic nucleus, which are greatly impli-
cated in modulating responses to stress (Spencer et al. 2004;
Adamec et al. 2010; Crawford et al. 2010), also displayed
neuronal activation after O3 inhalation. Worthy of note was
the activation of the CeA, a projection site of the NTS and
the external lateral PBN (Gaykema et al. 2007) known to be
more responsive to systemic stressors than to processive ones
that activate predominantly medial and basolateral amygdala
nuclei (Ulrich-Lai and Herman 2009). More particularly
interesting in this context was the observation that in the
BST, which is a limbic structure controlling vital homeostatic
functions and integrating stress-related behavioral functions,
 2011 The Authors
Journal of Neurochemistry  2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 961–972
14714159, 2011, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.2011.07267.x by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [04/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
only the lateral part that is closely related to CeA (Gaykema
et al. 2007; Ulrich-Lai and Herman 2009) was activated by
O3 challenge.
It therefore appears that O3 inhalation evoked a neuronal
activation pattern very similar to the one promoted by
systemic stress and different from the one elicited by
processive stress. Coherent with this view, O3 challenge
did not induce neuronal activation in limbic structures
associated with processive stress response, such as the
hippocampus and the medial prefrontal cortex.
Consequences of the activation of stress-related CNS
structures are not anodyne. Indeed, chronic stress is known to
result in disruption of sleep pattern, anxiety, depression and
social isolation (reviewed in Chrousos 2009). More specif-
ically, we highlighted neuronal activation in the LC and in
the DR that are involved in sleep pattern regulation (Kalia
2006), as well as in the CeA and in the BST that participate
in memory processing (Liang et al. 1983; Ledoux and Muller
1997). Altogether, these observations suggest that some
neurobehavioral disorders observed after O3 exposure could
result from selective sustained activation of stress-related
brain regions.
Conclusion
Our study is the first anatomical description of the CNS
regions activated by O3 inhalation in adult rats. It provides
evidence that O3 exposure activated the CNS through the
vagus nerves without the involvement of the thoracic spinal
nerves. The O3 challenge dramatically activated not only the
NTS but also several other brain structures belonging to
various stress-responsive regions, including brainstem, hypo-
thalamus and amygdala. This peculiar neuronal activation
footprint not only suggests that the brain interprets O3
exposure as a systemic stress, but could also explain some of
the CNS alterations encountered following O3 exposure.
Acknowledgement
This work was supported by Centre National de la Recherche
Scientifique (CNRS), Agence Française de Sécurité Sanitaire de
l’Environnement et du Travail (AFSSET, Grant EST-2008/1/47/
STRUBE), Agence De l’Environnement et de la Maı̂trise de
l’Energie and région Provence-Alpes-Côte d’Azur (ADEME/région
PACA, fellowship to LS). We gratefully acknowledge AtmoPACA
for its technical contribution. We wish to thank Pr. Y. Jammes for the
fruitful discussions and the technical help.
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