J Appl Physiol
91: 2157–2165, 2001.
Prenatal nicotine affects catecholamine gene expression
in newborn rat carotid body and petrosal ganglion
ESTELLE B. GAUDA, REED COOPER, PATRICE K. AKINS, AND GUIMEI WU
Department of Pediatrics, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287-3200
Received 29 March 2001; accepted in final form 25 June 2001
peripheral arterial chemoreceptors; tyrosine hydroxylase; dopamine ␤-hydroxylase; preproenkephalin; control of breathing; sudden infant death syndrome
the biological mechanisms that
help explain the increased association between sudden
infant death syndrome and exposure to tobacco smoke
(19), several physiology studies have investigated the
effect of prenatal nicotine exposure, a major component
of tobacco smoke, on ventilatory and cardiovascular
control in newborn animals. In newborn rats exposed
prenatally to nicotine, abnormalities in ventilation
(46), autoresuscitation (11), and cardiovascular responses (32) have been reported. Specifically, prenatal
exposure to nicotine blunts the ventilatory responses to
short exposures to hypoxia (46) and hyperoxia (2),
TO BETTER UNDERSTAND
Address for reprint requests and other correspondence: E. B.
Gauda, Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, Maryland 21287–3200 (E-mail: [email protected]).
http://www.jap.org
manipulations that are typically used as a test of
peripheral chemoreceptor function in unanesthetized
animals and newborn infants.
Nicotine binds to nicotinic cholinergic receptors on
catecholamine-containing neurons and affects neurotransmitter expression in the peripheral and central
nervous systems (31, 51). Nicotine increases dopamine,
norepinephrine, and opioid content and upregulates
catecholaminergic synthesizing enzymes and peptide
expression (8). The peripheral arterial chemoreceptors
express nicotinic receptors (1), contain catecholamines
and opioids, and are involved in modulating respiratory and arousal responses to hypoxia (for review, see
Ref. 20). In the peripheral arterial chemoreceptors,
catecholamines are the most abundant neuromodulators in the carotid body. Dopamine, through binding
to dopaminergic D2 receptors, and norepinephrine,
through binding to ␣2-adrenergic receptors on carotid
sinus nerve fibers, attenuate hypoxic chemosensitivity
(for review, see Ref. 20). Opioids, specifically, Metenkephalins through binding to ␦-opioid receptors, also
attenuate hypoxic chemosensitivity (35).
Hypoxic chemosensitivity of peripheral arterial chemoreceptors increases with postnatal age (for review,
see Refs. 17 and 10). Associated with maturation of
hypoxic chemosensitivity are changes in neurotransmitter profiles and changes in the biochemical and
electrical properties of cells in the carotid body (for
review, see Ref. 17). Specifically, dopamine content is
elevated in the carotid body of newborn rats, and this is
associated with blunted chemoreceptor responses (25).
Similarly, our laboratory has shown that mRNA levels
for tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine synthesis, are significantly elevated in newborn animals, whereas mRNA levels for
dopamine D2 receptors are decreased in the same cells
in the carotid body (14). However, with increasing
maturation, TH mRNA levels decreased, whereas dopamine D2-receptor mRNA levels increased. Less is
known about the effect of maturation on the enkephalin content in the carotid body. In addition, our laboratory has previously demonstrated that preproenkephalin (PPE) mRNA is present in petrosal ganglion
cells but not in the carotid body of 14-day-old rats (16).
The costs of publication of this article were defrayed in part by the
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solely to indicate this fact.
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society
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Gauda, Estelle B., Reed Cooper, Patrice K. Akins,
and Guimei Wu. Prenatal nicotine affects catecholamine
gene expression in newborn rat carotid body and petrosal
ganglion. J Appl Physiol 91: 2157–2165, 2001.—Nicotine
exposure modifies the expression of catecholamine and opioid
neurotransmitter systems involved in attenuation of hypoxic
chemosensitivity. We used in situ hybridization histochemistry to determine the effect of prenatal and early postnatal
nicotine exposure on tyrosine hydroxylase (TH), dopamine
␤-hydroxylase (D␤H), preproenkephalin (PPE), and D2-dopamine receptor mRNA levels in the rat carotid body and
petrosal ganglion during postnatal development. In the carotid body, nicotine increased TH mRNA expression in animals at 0 and 3 postnatal days (both, P ⬍ 0.05 vs. control)
without affecting TH mRNA levels at 6 and 15 days. At 15
postnatal days, D␤H mRNA levels were increased in the
carotid body of nicotine-exposed animals. Dopamine D2-receptor mRNA levels in the carotid body increased with postnatal age but were unaffected by nicotine exposure. PPE was
not expressed in the carotid body at any of the ages studied in
control or treated animals. In the petrosal ganglion, nicotine
increased the number of ganglion cells expressing TH mRNA
in animals at 3 days (P ⬍ 0.01 vs. control). D␤H mRNA
expression was not induced nor was PPE mRNA expression
increased in the petrosal ganglion in treated animals. Prenatal nicotine exposure upregulates mRNAs involved in the
synthesis of two inhibitory neuromodulators, dopamine and
norepinephrine, in peripheral arterial chemoreceptors, which
may contribute to abnormalities in cardiorespiratory control
observed in nicotine exposed animals.
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PRENATAL NICOTINE EXPOSURE MODIFIES GENE EXPRESSION
METHODS
Time-dated, pregnant Sprague-Dawley rats were used. On
days 2–3 of gestation, before implantation of the embryo in
the uterine wall, an osmotic minipump (type 2ML4, Alza,
Palo Alto, CA) containing nicotine bitartrate or sterile saline
(0.9%) was inserted subcutaneously. The dose of nicotine
delivered by the osmotic pump (6 mg 䡠 kg⫺1 䡠 day⫺1 of free base
nicotine) produces plasma nicotine levels that are seen in
heavy smokers (3 packs/day) (31, 38). Dams received nicotine
or vehicle until parturition on day 22 and then for 1 wk after
delivery, i.e., for a total of 4 wk. After spontaneous delivery,
the rat pups were cross-fostered to maintain equal litter
sizes.
Tissues were taken from Sprague-Dawley rats on postnatal days 0, 3, 6, and 15 (n ⫽ 7, each age). All animals were
briefly anesthetized with 3% methoxyflurane and decapitated. The bifurcation of the carotid artery with the carotid
body and the superior cervical, nodose, petrosal, and jugular
ganglia was quickly removed en bloc, placed in embedding
media (Fisher), and quickly frozen on dry ice. The right and
left tissue blocs were removed from the animals within 5 min
of decapitation. The tissues were stored at ⫺70°C until further processing.
In situ hybridization histochemistry. Tissue blocs were cut
in 12-␮m sections on a cryostat. Sections were thaw-mounted
onto gelatin-chrome, alum-subbed slides. Slide-mounted sections were then fixed in 4% paraformaldehyde, acetylated in
fresh 0.25% acetic anhydride in 0.1 M triethanolamine, dehydrated in ascending series of alcohols, delipidated in chloroform, and then rehydrated in a descending series of alcohols. Slides were air dried and then stored at ⫺20°C.
Antisense ribonucleotide probes were used for detection of
TH, D␤H, PPE, and dopamine D2-receptor mRNAs. The
antisense probes were constructed from cDNAs for each of
these genes by in vitro transcription. The cDNA for the TH,
PPE, and dopamine D2 receptors was complementary to base
pairs 1,120–1,488 (22), 51–987 (52), and 372–1,174 (3, 37) of
the rat genes, respectively. cDNA for D␤H contained a 575J Appl Physiol • VOL
base pair fragment corresponding to the nucleotides 205–780
of the rat D␤H cDNA described by McMahon et al. (34). The
cDNA fragment was obtained by PCR amplification of rat
striatum cDNA with sense strand oligonucleotide corresponding to base pairs 205–225 and antisense oligonucleotide strand corresponding to bases 759–780 of the published
sequence (34). The PCR products, along with control samples, were electrophoresed through an agarose gel. A single
band of ⬃500 base pairs was excised from the gel and subsequently subcloned into pCR4-TOPO TA cloning vector (Invitrogen, Carlsbad, CA). This cloning vector allows for direct
cloning of PCR products into an EcoR I (restriction enzyme)
cloning site and contains T3 and T7 RNA polymerase sites for
generating sense and antisense ribonucleotide probes for in
situ hybridization experiments and direct sequencing. The
subcloned cDNA fragment was partially sequenced to determine orientation of the cloned fragment.
To verify specificity of the ribonucleotide probes, coronal
brain sections from adult animals corresponding to the following bregma coordinates (bregma: ⫺10.04, ⫺5.80, and
0.20) (41) were used as controls. Control brain slides were
hybridized, exposed, and developed simultaneously with the
experimental slides. Uniformly, these control slides demonstrated the known patterns of gene expression in striatopallidal neurons for dopamine D2-receptor and (18) PPE mRNAs
(44), nigrostriatal neurons for TH (40), and locus coeruleus
neurons for D␤H (data not shown). All control brain slides
showed discrete and specific hybridization signal in the neural cell groups known to express these genes without any
background or nonspecific hybridization signal. In addition,
the patterns of TH, PPE, D␤H, and dopamine D2-receptor
mRNA expression differed in the tissue bloc of the carotid
body and petrosal and superior cervical ganglia, further
demonstrating probe specificity.
Probes were labeled with [35S]UTP via in vitro transcription as outlined by Chesselet et al. (5). Labeled probes of
1.2–1.5 ⫻ 106 dpm were added to 100 ␮l of hybridization
buffer [50% formamide, 600 mM NaCl, 300 mM NaCl, 20 mM
Tris 䡠 HCl, pH 7.5, 1 mM EDTA, 10% dextran sulfate, 1⫻
Denhardt’s solution, 100 ␮g/ml salmon sperm DNA, 250
␮g/ml yeast total RNA (type XI), 250 ␮g/ml yeast tRNA, and
100 mM dithiothreitol], which was applied to slides containing 8–10 sections per slide. Hybridization was performed at
55°C overnight. The slides were then washed in 1⫻ SSC (0.15
M sodium chloride-0.015 M sodium citrate, pH 7.2) at room
temperature. After treatment with RNAase A (20 mg/ml),
slides were washed at 60°C in 0.2⫻ SSC, rinsed in deionized
water, and air dried. Slides were then dipped in Kodak
photographic emulsion, dried, and exposed in the dark at
⫺20°C for 4–8 wk. After exposure, the slides were thawed at
room temperature, developed with Dektol (Kodak), and counterstained with thionin, and coverslips were applied with
Permount.
Data analysis. Comparisons were only made between data
obtained from slides that were processed for in situ hybridization, hybridized, exposed, and developed together under
the same conditions. Silver grains generated by 35S in the
emulsion were analyzed by using a microscope and Macintosh image analysis program [National Institutes of Health
(NIH) image, W. Rasband, NIH]. Semiquantitation of silver
grains (representing TH, D␤H, PPE, and dopamine D2-receptor mRNAs) in the carotid body was done by using a counting
algorithm of the image analysis program. After the dark-field
image was captured and digitized at ⫻400, the number of
grains was counted in the carotid body. To normalize for the
possible different sizes of the sections of the carotid bodies,
the number of grains in multiple, contiguous 100-␮m-diam-
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Acute nicotine exposure affects catecholaminergic and
opioid neurotransmitters in peripheral and central
nervous systems. However, how prenatal nicotine exposure affects the expression of these inhibitory neurotransmitter systems in the carotid body and sensory
ganglion is not known. Thus the purpose of this study
was to elucidate molecular mechanisms involved in
alterations in the expression of critical neurotransmitters that may explain alterations in chemosensitivity
observed in newborn rats exposed prenatally to nicotine. We determined the effect of prenatal and early
postnatal nicotine exposure on TH, PPE, and dopamine
D2-receptor gene expression in the carotid body and
petrosal ganglion in newborn rat pups during the first
2 wk of postnatal development. We also determined the
effect of prenatal nicotine exposure on dopamine ␤-hydroxylase (D␤H) gene expression in the carotid body
and petrosal ganglion in animals at 2 wk postnatal age.
Because nicotine exposure upregulates catecholaminergic and opioid traits in central and peripheral nervous systems, we hypothesized that prenatal nicotine
exposure would affect neurotransmitters by upregulating mRNAs encoding proteins involved in inhibitory
catecholaminergic and opioid systems in peripheral
arterial chemoreceptors during early postnatal ages.
PRENATAL NICOTINE EXPOSURE MODIFIES GENE EXPRESSION
eter circles was counted, and a mean was obtained for each
section of the carotid body. These numbers were combined
from two to three sections of the carotid body to obtain an
average count per animal. The population of values of average counts per animal was compared between control and
treated groups with ANOVA and post hoc analysis via Student’s unpaired t-test. Statistical significance was set at P ⬍
0.05. Because of the additional tissue sections that were
available from 15-day-old animals, we determined whether
prenatal nicotine exposure altered D␤H gene expression in
the carotid body and petrosal ganglion in this age only.
Semiquantitation of the number of ganglion cells in the
petrosal/jugular ganglion (PG/JG) complex expressing TH
mRNA was determined by counting the number of positive
cells per tissue section with dark-field microscopy at ⫻400. A
mean was obtain from each animal after determining the
mean number of TH-positive ganglion cells from five to seven
tissue sections per animal. The mean number of TH-positive
ganglion cells per PG/JG section between control and nicotine-exposed animals was determined by unpaired t-test in
animals at 3 and 15 postnatal days of age.
Prenatal nicotine exposure did not affect litter sizes
nor did it affect dam or rat pup mortality. However, rat
pups exposed to nicotine were smaller than control
pups at each of the postnatal ages studied (Table 1).
Prenatal and early postnatal nicotine exposure significantly increased the expression of TH mRNA in the
carotid bodies of newborn rats (P ⫽ 0.02, ANOVA). TH
mRNA levels were greater in animals exposed to nicotine at 0 and 3 postnatal days, as shown in representative photomicrographs from four animals in Fig. 1
and in the histogram of composite data from all animals in Fig. 2. Although TH mRNA levels at days 6 and
15 did not differ between control and treated animals
(Fig. 2), nicotine exposure did upregulate D␤H mRNA
expression in the carotid body of 15-day-old animals
(P ⫽ 0.03, control vs. nicotine), as shown in the photomicrographs of one control and one prenatally nicotine-exposed animal (Fig. 3, A and B), along with composite data (bar graph) from all animals (Fig. 3, right).
Similar to TH mRNA, D␤H mRNA was moderately
expressed in many cells in the superior cervical ganglion in control animals, as shown in the low- (Fig. 4A)
and high-power (Fig. 4B) dark-field photomicrographs
from one control animal at day 15. However, prenatal
nicotine exposure did not significantly change the level
of D␤H mRNA levels in the superior cervical ganglion
(data not shown). Prenatal nicotine exposure also increased the number of ganglion cells in the PG/JG
Table 1. Effect of prenatal nicotine exposure
on body weight
Postnatal
Day
Nicotine, g
Control, g
P Value
0
3
6
15
5.7 ⫾ 0.3
7.8 ⫾ 0.5
13.9 ⫾ 0.7
34.2 ⫾ 1.4
6.1 ⫾ 0.2
9.1 ⫾ 0.4
14.8 ⫾ 0.9
39.6 ⫾ 0.9
⬍0.05
⬍0.05
⬍0.05
⬍0.05
Values are means ⫾ SD; n ⫽ 7 at each age.
J Appl Physiol • VOL
complex expressing TH mRNA at 3 days (Fig. 5) but
not at 15 postnatal days (P ⫽ 0.2, control vs. nicotine at
3 days). D␤H mRNA was not detected in the PG/JG
complex in either control or treated animals at day 15.
Dopamine D2-receptor mRNA was expressed in the
petrosal ganglion and carotid body in both control and
treated animals. The low-power, bright-field photomicrograph of the nissl-stained tissue section (Fig. 6A)
depicts the anatomy of the petrosal ganglion, IX cranial nerve, carotid sinus nerve, and carotid body. The
high-power, dark-field photomicrograph (Fig. 6B) of
the same tissue section shown in Fig. 6A shows clusters of silver grains representing dopamine D2-receptor
mRNA within ganglion cells of the petrosal ganglion.
As previously described (14) and shown in this study,
dopamine D2-receptor mRNA levels in the carotid body
increased with postnatal age. However, nicotine exposure did not alter the level of dopamine D2 expression
in the carotid body or the petrosal ganglion at any of
the four postnatal ages studied (Fig. 7).
PPE mRNA was not expressed in the carotid body at
any of the ages studied in either control or nicotineexposed animals. Although PPE was not expressed in
the carotid body, it was expressed in the superior
cervical and petrosal ganglia as previously described
(16). Nicotine exposure did not induce PPE gene expression in the carotid body nor did it change PPE
mRNA levels in the superior cervical or petrosal ganglia (data not shown).
DISCUSSION
Experimental models investigating neurotransmitter expression in the peripheral and central nervous
system strongly suggest that alterations in cholinergic
neurotransmission affect neurotransmitter phenotypes in immature and mature neurons (30, 31, 39, 51).
This study is the first to show that continuous prenatal
nicotine exposure during fetal development and early
postnatal exposure modifies catecholaminergic gene
expression in the peripheral arterial chemoreceptors.
Nicotine exposure increases TH mRNA expression in
both the carotid body and PG/JG complex in animals
within the first 3 days of postnatal life and upregulates
D␤H mRNA levels in the carotid body of 15-day-old
animals. However, prenatal nicotine exposure does not
alter dopamine D2-receptor mRNA levels in the carotid
body or petrosal ganglion. In addition, we have shown
that PPE mRNA is not expressed in the carotid body
but is expressed in the superior cervical and petrosal
ganglion, and the level of expression is not altered by
prenatal exposure to nicotine. Thus alterations in transynaptic neurotransmission affected by prenatal and
early postnatal nicotine exposure induce catecholaminergic neurotransmitter expression in peripheral arterial chemoreceptors in newborn rat pups.
We observed an increase in TH mRNA expression in
the carotid body of newborn animals at 0 and 3 postnatal days and in the petrosal ganglion in 3-day-old
animals exposed to nicotine. However, this effect was
no longer seen at 6 and 15 postnatal days. The most
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RESULTS
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PRENATAL NICOTINE EXPOSURE MODIFIES GENE EXPRESSION
Fig. 1. Dark-field photomicrographs of the carotid body
showing the expression of tyrosine hydroxylase (TH)
mRNAs in control animals at 0 (A) and 3 postnatal days
(C) and animals treated prenatally with nicotine at 0
(B) and 3 postnatal days (D). Nicotine exposure increased the expression of TH mRNA in the carotid body
in animals at 0 and 3 postnatal days (B and D). Silver
grains appear as white dots. Scale bars ⫽ 25 ␮m.
Fig. 2. Bar graph showing TH mRNA levels in the carotid body
during postnatal development in control animals (open bars) and
animals treated prenatally with nicotine (solid bars). Values are
means ⫾ SE; n ⫽ 7 (ANOVA, P ⫽ 0.02). Significant difference
(Student’s unpaired t-test): * P ⫽ 0.02 vs. control at day 0; ⫹ P ⫽
0.002 vs. control at day 3.
J Appl Physiol • VOL
tact peripheral chemoreceptors at birth appear to provide essential trophic influences for maintaining
rhythmic breathing during maturation (for review, see
Ref. 17). Animals exposed prenatally to nicotine have
1) reduced ventilation during air breathing and in
response to hypoxia (46), 2) less reduction in ventilation in response to hyperoxia (test of peripheral chemoreceptor function) (2), 3) reduced ability to autoresuscitate from repeated episodes of hypoxia (11), and 4)
increased mortality from severe, continuous hypoxic
exposure (45). Alterations in these cardiorespiratory
reflexes may involve adverse molecular and cellular
changes in peripheral arterial chemoreceptors induced
by prenatal nicotine exposure during prenatal development. Bamford and Carroll (2) reported altered peripheral chemoreceptor function (Dejours’ test) in awake
3-day-old newborn animals that were exposed prenatally to nicotine. However, these investigators, using a
superfused ex vivo preparation, were unable to detect a
difference between control and treated animals when
measuring activity from the whole carotid sinus nerve
in response to a severe hypoxic challenge (5% O2-5%
CO2). Yet the preponderance of physiological studies
suggest that peripheral arterial chemoreceptor responses could be affected by prenatal exposure to nicotine. One explanation for the observed discrepancy
between the whole animal and the isolated carotid
body preparations might be secondary to the isolated
preparation used. The severe hypoxia stimulus may
have saturated the whole nerve response, making it
difficult to distinguish differences in the carotid sinus
nerve activity in the preparations between control and
prenatally treated animals. Our study is the first to
demonstrate that changes in gene expression in the
peripheral chemoreceptors are induced by prenatal
and perinatal exposure to nicotine. Increased expression of inhibitory catecholaminergic traits may in part
explain the alterations in cardiorespiratory responses
observed in animals treated prenatally with nicotine.
Nicotine exposure affects catecholaminergic traits in
the peripheral and central nervous system: comparison
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plausible explanation for the loss of effect is that the 6and 15-day-old animals were no longer being exposed
to nicotine. The mini-osmotic pump containing nicotine
or sterile saline was not removed from the nursing
dam, and the pump delivered nicotine or sterile water
for ⬃1 wk postpartum. The 6-day-old animals may
have received some nicotine via the milk; however, the
relative dose per weight was considerably less than
that of 0- and 3-day-old animals.
Cardiorespiratory physiology is altered in rats exposed prenatally to nicotine. Peripheral chemoreceptors are involved in multiple cardiorespiratory functions that include, but are not limited to, modulating
respiration in response to changes in temperature,
chemical stimuli, and recovery from asphyxial apnea
associated with upper airway obstruction (12). Intact
peripheral chemoreceptors are not essential for establishment of rhythmic breathing at birth. However, in-
PRENATAL NICOTINE EXPOSURE MODIFIES GENE EXPRESSION
2161
to and extension of previous literature. Acute nicotine
exposure induces upregulation of catecholaminergic
synthesizing enzymes in several dopaminergic and
noradrenergic cell groups in the central and peripheral
nervous system (43, 47). Furthermore, nicotine significantly increases TH mRNA levels in PC-12 cells (27),
an immortalized cell line that is frequently used as a
model of chemosensitive type I cells in the carotid body
(36). Type I cells in the carotid body contain TH, dopamine, and norepinephrine, and acute nicotine exposure
increases dopamine and norepinephrine release from
dissociated type I cells (48). In 3-day-old rat pups,
acute postnatal nicotine exposure reduced dopamine
content and increased TH mRNA expression in the
Fig. 4. Representative low- (A) and high-power (B) dark-field photomicrograph showing the specificity and cellular localization of D␤H
mRNA expression in the superior cervical ganglion (SCG) of a 15day-old animal. Scale bars ⫽ 50 ␮m.
J Appl Physiol • VOL
carotid bodies (28). Furthermore, chemoreceptor function was diminished in these 3-day-old rat pups treated
acutely with nicotine (28). We have extended the findings of Holgert et al. (28) by showing that chronic
prenatal and early postnatal exposure to nicotine is
associated with increased TH mRNA expression in the
carotid body and petrosal ganglion of newborn rat pups
at 0 and 3 postnatal days. Although the effect of nicotine exposure on TH mRNA levels in the carotid body of
15-day-old animals was no longer apparent, we did
observe an increase in D␤H gene expression in the
carotid body in these animals. Because of the limitation of available tissue, we do not know if nicotine
exposure also increased D␤H mRNA levels in the
younger age groups. Similar to the findings of Holgert
et al., we report no significant effect of nicotine exposure on dopamine D2-receptor mRNA levels in the
carotid body.
Possible mechanisms for nicotine-induced upregulation of TH and D␤H mRNAs in peripheral chemoreceptors. Several mechanisms, either indirectly or directly,
may account for the upregulation of TH mRNA levels
in the carotid body and petrosal ganglion induced by
prenatal nicotine. The most plausible indirect mechanism is by inducing hypoxemia in the fetus. It is possible that prenatal nicotine exposure in the dose used
in this experiment may have induced vasoconstriction
of uterine vessels with subsequent decreased oxygen
delivery to the rat pups. The rat pups exposed prenatally to nicotine were smaller than the control animals,
an observation that has also been reported by other
investigators using the same paradigm of nicotine exposure (2, 45). Litter sizes were matched between nicotine and control animals, although our animals were
not pair-fed. We chose the dose of nicotine used in this
study because equivalent dosing and exposure para-
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Fig. 3. Photomicrographs (A and B) and bar graph (right) showing effect of prenatal nicotine exposure on dopamine
␤-hydroxylase (D␤H) mRNA expression in the carotid body of 15-day-old animals. Representative photomicrographs of a dark-field image of the carotid body (CB) in two 15-day-old animals show the expression of D␤H mRNA
in a control animal (A) and an animal treated prenatally with nicotine (B). Nicotine exposure increased the
expression of D␤H mRNA in the carotid body. Arrows are pointing to clusters of silver grains that represent D␤H
mRNA expression. Scale bars ⫽ 25 ␮m. Right: bar graph showing D␤H mRNA levels in the carotid body control
animals (open bar) and animals treated prenatally with nicotine (solid bar) at 15 days postnatal age. Values are
means ⫾ SE; n ⫽ 5. * P ⫽ 0.02 vs. control.
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PRENATAL NICOTINE EXPOSURE MODIFIES GENE EXPRESSION
digms have been used in studies showing abnormalities in hypoxic or hyperoxic chemosensitivity in newborn rat pups (2, 45, 46). Nevertheless, transcription,
regulation, and enzymatic function of TH are significantly altered by the partial pressure of oxygen. TH
gene expression within 1 h of hypoxic exposure is
induced in the carotid body of adult rats (6). The
increase in TH mRNA levels appears to be secondary to
an increase in the transcription and stabilization of the
mRNA transcripts, as suggested by experiments exposing PC-12 cells to hypoxia (7). Although our laboratory
has also shown that hypoxia significantly upregulates
TH mRNA levels in the carotid body, it did not increase
the grains per cell or the number of cells expressing TH
mRNA in the PG/JG ganglion complex (15). In contrast, in our present study, prenatal exposure to nicotine did increase the number of ganglion cells expressing TH mRNA.
Alternatively, nicotine may have directly increased
TH and D␤H mRNA levels in the carotid body and
petrosal ganglion by a cascade of intracellular events,
thus leading to an increase in gene transcription similar to its effects in PC-12 cells (23) and in the rat
Fig. 6. Photomicrographs [bright-field (A) and dark-field (B)] of
nissl-stained tissue section showing the histology of the PG, IX
cranial nerve (CN), carotid sinus nerve (CSN), and the CB (A) and
the expression of dopamine D2-receptor (D2-R) mRNA in the PG (B)
of a 6-day-old animal. Silver grains are clusters of white dots (arrows). Scale bars ⫽ 100 ␮m.
Fig. 7. Bar graph showing dopamine D2-receptor mRNA levels
(grains/area) in the carotid body during postnatal development in
control animals (open bars) and animals treated prenatally with
nicotine (solid bars). Nicotine exposure did not change the level of
dopamine D2-receptor mRNA levels in the carotid body during the
first 2 wk of postnatal development. Values are means ⫾ SE; n ⫽ 7.
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Fig. 5. Photomicrographs (A and B) and bar graph (right) showing effect of prenatal nicotine exposure on TH
mRNA expression in the petrosal ganglion (PG), jugular ganglion (JG), and SCG of 3-day-old animals. Representative low-power, dark-field photomicrographs are shown of PG, JG, and SCG of 3-day-old animals, showing the
increase in PG/JG neurons expressing TH mRNA (arrows) in an animal exposed prenatally to nicotine (B)
compared with the control animal (A). Silver grains are clusters of white dots (arrows). Scale bars ⫽ 50 ␮m. Right:
bar graph showing the effect of prenatal nicotine exposure on the no. of ganglion cells in the PG/JG complex
expressing TH mRNA in 3-day-old animals. Open bar, control animals; solid bar, animals exposed prenatally to
nicotine. Values are means ⫾ SE; n ⫽ 5 each group. * P ⬍ 0.01 vs. control at 3 days.
PRENATAL NICOTINE EXPOSURE MODIFIES GENE EXPRESSION
J Appl Physiol • VOL
been localized to nerve fibers innervating the carotid
body (26). Our previous and present findings show that
PPE mRNA was abundantly expressed in the cell bodies in the petrosal ganglion but was not detected in the
carotid body, corroborating the immunohistochemical
studies in the rat. Similar cellular mechanisms are
involved in the nicotine-induced upregulation of PPE
gene transcription as they are in TH and D␤H gene
transcription. PPE mRNA is present in the striatum
and adrenal chromaffin cells, and acute nicotine induces the expression of PPE in bovine adrenal chromaffin cells in culture (49) and in the striatum (9, 29)
and hippocampus of adult rats (29). However, prenatal
exposure to nicotine at the dose used in this study did
not induce PPE gene expression in the carotid body nor
does it upregulate PPE mRNA in the cell bodies of the
petrosal ganglion. Similar to our findings, chronic vs.
acute nicotine exposure does not upregulate PPE
mRNA expression in the striatum or hippocampus of
the adult rat (30).
In summary, prenatal nicotine exposure is associated with blunted peripheral chemoreceptor function
(46) and reduced ability to autoresuscitate (11) in newborn animals. We have found that prenatal nicotine
exposure upregulates TH mRNA levels in both the
carotid body and petrosal ganglion in rat pups at 0 and
3 postnatal days and upregulates D␤H mRNA levels in
the carotid body of 15-day-old animals. However, dopamine D2-receptor and PPE mRNA levels in the carotid body or petrosal ganglion were unaltered in animals exposed prenatally to nicotine during the first 2
wk of postnatal life. TH and D␤H are the rate-limiting
enzymes for catecholamine and norepinephrine synthesis, respectively. Dopamine and norepinephrine,
through binding to inhibitory dopamine D2- and ␣2adrenergic receptors, respectively, on postsynaptic receptors within the carotid body, are associated with
reduced hypoxic chemosensitivity. Our data support a
possible role for upregulation of catecholamines in peripheral arterial chemoreceptors as one possible cellular mechanism that could explain the reduced physiological responses involving peripheral chemoreceptors
in newborn rat pups exposed prenatally to nicotine.
The authors thank Dr. Musa Haxhiu for reviewing the manuscript.
E. B. Gauda is a recipient of National Institute on Drug Abuse
Grant R01 DA-13940, which supported this work.
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