Neuroscience Letters 545 (2013) 102–106
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet
Brain nitric oxide metabolites in rats preselected for nicotine preference and
intake
Aysegul Keser a,b , Tanseli Nesil a,1 , Lutfiye Kanit a,b , Sakire Pogun a,∗
a
b
Ege University, Center for Brain Research, Bornova, Izmir, Turkey
Ege University, School of Medicine, Department of Physiology, Bornova, Izmir, Turkey
h i g h l i g h t s
•
•
•
•
•
Rats were exposed to oral nicotine choice starting at adolescence or adulthood.
Brain nitrate + nitrite determined in rats with maximum/minimum nicotine preference.
Control rats received only water from both bottles, naïve rats were group housed.
Isolation stress increased NO metabolites in the hippocampus (naïve–control).
Preference had no effect; adolescent-onset rats had higher NO activity in frontal cortex.
a r t i c l e
i n f o
Article history:
Received 19 February 2013
Received in revised form 5 April 2013
Accepted 14 April 2013
Keywords:
Nitric oxide
Preselected rats
Oral nicotine
Two-bottle free choice
Stress
a b s t r a c t
Nicotine addiction is a serious health problem resulting in millions of preventable deaths worldwide. The
gas messenger molecule nitric oxide (NO) plays a critical role in addiction, and nicotine increases nitric
oxide metabolites (NOx) in the brain. Understanding the factors which underlie individual differences in
nicotine preference and intake is important for developing effective therapeutic strategies for smoking
cessation. The present study aimed to assess NO activity, by measuring its stable metabolites, in three
brain regions that express high levels of nicotinic acetylcholine receptors in rats preselected for nicotine
preference. Rats (n = 88) were exposed to two-bottle, free choice of oral nicotine/water starting either
as adolescents or adults; control animals received only water under identical conditions. Following 12
or six weeks of exposure, levels of NOx (nitrite + nitrate), were determined in the hippocampus, frontal
cortex, and amygdala. Since the rats were singly housed during oral nicotine treatment, naïve rats were
also included in the study to evaluate the effect of isolation stress. Isolation stress increased NOx in the
hippocampus. Nicotine preference did not have a significant effect on NO activity, but rats with adolescent
exposure had higher NOx levels in the frontal cortex compared to adult-onset rats. Our findings suggest
that nicotine exposure during adolescence, regardless of the amount of nicotine consumed, results in
higher NO activity in the frontal cortex of rats, which persists through adulthood.
© 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Nitric oxide (NO) is a gaseous biological messenger molecule in
the central nervous system (CNS), which is synthesized by various
isoforms of nitric oxide synthase (NOS) that catalyze the conversion
of arginine to citrulline and NO [4,5]. Calcium/calmodulin is needed
for the activation of NOS, and Ca2+ influx is at least partly caused
by the stimulation of glutamate receptors. However, the activation
∗ Corresponding author at: Ege University, Center for Brain Research, Bornova,
35100 Izmir, Turkey. Tel.: +90 232 3882868; fax: +90 232 3746597.
E-mail addresses: [email protected], [email protected] (S. Pogun).
1
Current address: Department of Psychiatry and Neurobehavioral Sciences,
University of Virginia, Charlottesville, VA, USA.
0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neulet.2013.04.027
of other ion channels, such as the nicotinic acetylcholine receptor,
has also been shown to result in increased NO levels in the brain
[26]. NO regulates neurotransmitter uptake and mediates in the
neurotoxic actions of glutamate, as well as in learning and memory
processes [27].
The nitrergic system is also suggested to play a critical role
in addiction (see for examples [1,20,22,29,32]). In rats, NOS inhibition attenuates nicotine abstinence syndrome, suggesting the
involvement of NO in nicotine dependence [19]. We have shown
that acute and repeated nicotine administration increases levels
of the stable NO metabolites nitrite and nitrate (collectively, NOx)
in rat brain and that this effect is region-specific [26]. A followup study confirmed our results regarding increased NOx following
repeated nicotine administration; however, nicotine treatment
did not affect the number of NOS/nicotinamide dinucleotide
A. Keser et al. / Neuroscience Letters 545 (2013) 102–106
phosphate (NADPH)-diaphorase-positive NO-synthesizing neurons [36]. Inhaled NO from cigarette smoke is also suggested to
contribute to nicotine addiction in smokers [35].
Oral nicotine self-administration to rats, using a two-bottle free
choice design, mimics the human smoking condition by providing
rats with a choice of nicotine or water for 24 h/day for extended
periods. In this paradigm the animals have the opportunity to dose
their nicotine intake, based on their preferences [6]. In the present
study, rats were preselected based on their oral nicotine intake as
maximum and minimum nicotine preferring rats. Since the starting age of smoking varies in smokers, we used two groups of rats
with different ages at onset of nicotine treatment. Sex was included
as a factor because sex differences in the central effects of nicotine
and nicotine/tobacco dependence are well documented (reviewed
in [25,28]). The aim of the study was to assess NO activity, by
measuring its stable metabolites, in selected brain regions that
express high levels of nicotinic acetylcholine (ACh) receptors [34] in
rats with different preferences and different durations of nicotine
exposure.
2. Materials and methods
2.1. Animals
Male and female adult Sprague Dawley rats (n = 88), obtained
from Ege University Laboratory Animal Breeding Facility, were used
in the experiments. All animals were kept under standard laboratory conditions (20–22 ◦ C, humidity: 45–65%), were maintained
on a 12:12 h light:dark cycle (lights on 07:00–19:00), and fed ad
libitum (standard rat food pellets).
The animals were treated under the prescriptions for animal
care and experimentation of the pertinent European Communities
Council Directive of 24 November 1986 (86/609/EEC). The Institutional Animal Ethics Committee of Ege University approved all the
procedures.
2.2. Data collection and selection of animals
Three different groups of male and female rats were used in the
study.
(a) Naïve (four months of age): housed in same-sex groups, 3–4
animals/cage and received tap water from one bottle.
(b) Adolescent onset: housed singly after weaning (four weeks of
age). One week after acclimatization to single housing, rats were
given a free choice of oral nicotine (10 mg/L for two weeks,
20 mg/L for the remaining period)/water (n = 87; 41 male) or
water/water (controls, n = 13; 7 male) for twelve weeks, both
containing saccharine (10 g/L).
(c) Adult onset: housed singly after 13 weeks of age. One week
after acclimatization to single housing, rats were given a free
choice of oral nicotine (10 mg/L for two weeks, 20 mg/L for the
remaining period)/water (n = 67; 27 male) or water/water (controls, n = 23; 12 male) for six weeks, both containing saccharine
(10 g/L).
The protocol used in the present study was reported recently
[6,23]. Following oral nicotine exposure, rats were divided
into three different groups (Ward method, p < 0.01: maximum,
median and minimum nicotine preferring rats) according to
their average nicotine consumption (mg/kg/week) over 6 weeks
(weeks 1–6 in adult onset, and weeks 7–12 in adolescent onset
rats). Maximum and minimum nicotine preferring rats were
used in the experiments. Nicotine intake values are given in
Table 1.
103
Table 1
Nicotine consumption of maximum and minimum nicotine preferring rats.
MAXIMUM
MINIMUM
ADOLESCENT ONSET
MALE
FEMALE
8.68 ± 0.60 (n = 8) ###
8.49 ± 0.84 (n = 9) ##
1.23 ± 0.12 (n = 8) ***
1.99 ± 0.20 (n = 9) ***
ADULT ONSET
MALE
FEMALE
4.34 ± 0.84 (n = 6)
6.13 ± 1.05 (n = 6)
1.94 ± 0.21 (n = 6) **
2.17 ± 0.26 (n + 6) *
Rats were exposed to oral nicotine starting at the age of five (adolescent onset)
or twelve (adult onset) weeks of age. Average nicotine intake during six weeks as
adults (mg/kg body weight/week) ± SEM are given; the number of animals/group are
in parentheses. One-way ANOVA with eight groups revealed a significant difference
between the groups [F(7,53) = 20.09, p < 0.0001]. The results of post hoc LSD are:
different from MAXIMUM preferring, same sex *** p < 0.001, ** p < 0.01; different from
ADULT ONSET, same preference ### p < 0.001, ## p < 0.01.
2.3. Chemicals
Nicotine [(−) nicotine hydrogen tartrate]; nicotinamide dinucleotide phosphate (NADPH) (N9125), flavin adenine dinucleotide
(F2253), sulphanilamide (S9251) and N-1 naphtylethylenediamine
(N9125) were purchased from Sigma. Nitrate reductase from
Aspergillus sp. was obtained from Roche (10981249001).
2.4. Tissue preparation
Rats were taken from their home cages where the nicotine/water choice was available and were decapitated without
delay. Brains were rapidly removed and dissected (frontal cortex,
hippocampus, and amygdala) on ice.
2.5. Nitrite and nitrate determination
Brain nitrate was determined after its reduction to nitrite,
followed by the Griess reaction. Tissue samples were homogenized in phosphate buffer (pH 7.5) and centrifuged at 2000 × g
for 5 min. From the supernatant, 0.25 ml was diluted 4 times
and added to 0.25 ml of 0.3 M NaOH. After incubation for 5 min
at room temperature, 0.25 ml of 5% (w/v) ZnSO4 was added for
deproteinization. This mixture was then centrifuged at 3000 × g
for 20 min and the supernatant was used for the assays. Total
NOx levels in tissue homogenates were determined spectrophotometrically, based on the reduction of nitrate to nitrite by nitrate
reductase (EC 1.6.6.2) from Aspergillus sp. in the presence of
NADPH.
Sodium nitrate solutions were used for standard measurements. NOx levels in the tissues were expressed as mmol/g
wet weight. Protein concentrations were determined by Bradford
assay.
2.6. Statistical analyses
SPSS (v17.0) software was used for all statistical analyses. Data
was analyzed by ANOVAs; factors and dependent variables are
given in the Section 3. Post hoc tests were used to compare groups.
3. Results
The differences in the nicotine consumption of the rats
were analyzed by ANOVA with sex (male, female), preference
(maximum, minimum), and age at onset (adolescent, adult)
as factors and average nicotine consumption as the dependent variable. Preference, as expected, emerged as a significant
main effect [F(1,72) = 428.334, p < 0.0001] and interacted with age
[F(1,72) = 20.181, p < 0.0001]. Maximum nicotine preferring rats
drank significantly more nicotine solution than minimum preferring rats; and although the adolescent-onset maximum preferring
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A. Keser et al. / Neuroscience Letters 545 (2013) 102–106
Fig. 1. NOx (nitrite + nitrate) levels the hippocampus, frontal cortex and amygdala
of naïve (group housed) and control (singly housed) male and female rats. The bars
show average ± sem, different from males **p < 0.01, *p < 0.05, t-test.
rats consumed more nicotine than adult-onset maximum preferring rats, age at onset did not affect nicotine consumption in
minimum preferring groups (Table 1).
Since the rats were singly housed during the two-bottle free
choice oral nicotine treatment, naïve rats and control animals were
compared to assess the possible effect of single vs. group housing. NOx levels did not differ between brain regions of control
rats in the adolescent- and adult-onset groups. Therefore, all the
control groups were pooled and compared with the NOx levels of
naïve rats. ANOVAs with sex and single housing (naive, control) as
the factors revealed a main effect of housing in the hippocampus
[F(1,36) = 12.646, p = 0.001]; NOx levels were higher in control rats
than naïve animals (Fig. 1).
NOx levels in the control and experimental groups in each
brain region are shown in Fig. 2, panels (a), (b) and (c). To
assess the effect of nicotine treatment, we performed a series of
ANOVAs. First we did a repeated measures ANOVA with brain
region (amygdala, frontal cortex, hippocampus) as the withinsubjects factor; age at onset (adult, adolescent), sex (male, female),
and preference (control, minimum, maximum) as the betweensubjects factors; and NOx levels as the dependent variable. A
significant effect of brain regions emerged and interacted with
age at onset [F(2,130) = 15.965, p < 0.0001]. Overall, the highest
levels were in the hippocampus, followed by frontal cortex and
amygdala. While this pattern was seen in the rats exposed to
nicotine as adolescents, the rats exposed to nicotine as adults
still had the highest levels of NOx in the hippocampus, but
levels were higher in the amygdala than in the frontal cortex.
When we analyzed our results for adult-onset and adolescentonset rats separately, again, the only significant effect was region:
[F(2,32) = 14.576, p < 0.0001] and [F(2,98) = 31.787, p < 0.0001] for
adult and adolescent exposure, respectively. Since region emerged
as a significant main effect, we continued our analyses for each
region separately. A significant effect of age at onset was found
the in the amygdala [F(1,52) = 8.105, p = 0.007] and frontal cortex
[F(1,52) = 50.847, p < 0.0001]; in the amygdala, levels in adultonset rats were higher than adolescent-onset, while in the frontal
cortex levels were lower in the adult-onset rats than adolescentonset.
To compare the age and sex effects for each group separately
(control, minimum and maximum preferring rats), we performed
two-way ANOVAs. There was no effect observed in the control
groups. Both in the minimum [F(1,29) = 23.207, p < 0.0001] and
maximum [F(1,2) = 32.351, p < 0.0001] nicotine preferring rats, a
Fig. 2. NOx levels in the (a) hippocampus, (b) frontal cortex and (c) amygdala of male
and female rats which received a choice of water/water (control) or nicotine/water
(minimum: MIN or maximum: MAX). The MIN group was selected for low preference
and the MAX group for high preference for the nicotine solution. Nicotine exposure
started at 5 weeks of age in the adolescent- and at 14 weeks of age in the adult-onset
rats. ANOVA revealed a significant effect of age at onset in the frontal cortex with
adolescent-onset rats having higher levels of NOx than adult-onset rats.
main effect of age at onset was observed in the frontal cortex;
adolescent-onset rats had higher NOx levels than adult-onset animals. In the amygdala, the reverse trend was seen in age effect
in both minimum and maximum nicotine preferring rats, but it
was not significant: [F(1,29) = 4.006, p = 0.056] and [F(1,22) = 4.088,
p = 0.057], respectively.
A. Keser et al. / Neuroscience Letters 545 (2013) 102–106
4. Discussion
When designing this study, we hypothesized that in rats with
maximum preference for nicotine, brain levels of NO metabolites
will be higher than in those with minimum preference and that
there will be sex and regional differences. While our data clearly
show that there are regional differences in NOx levels, we did not
observe a significant effect of nicotine preference or sex following long-term nicotine intake. On the other hand, age at onset of
nicotine exposure did have an effect on NO activity in the frontal
cortex.
Single housing can be a stress factor, namely isolation stress, in
oral nicotine intake experiments. In the present study we observed
a main effect of housing on nitric oxide metabolites in the hippocampus; levels were significantly higher in the singly housed
control rats compared to naïve animals housed 3–4 same sex animals per cage. Similar to our findings, chronic isolation stress is
reported to increase NO metabolites in the hippocampus of male
Wistar rats, while no significant effect was observed in the prefrontal cortex [38]. We have previously shown that chronic saline
injections increased NO metabolites in the hippocampus in Sprague
Dawley rats [26]. The regulation of neuronal nitric oxide synthase
(nNOS) activity and expression in the rodent hippocampus by psychological stress has been shown in many studies [9,11,17,18,37].
We have recently shown that forced swim stress and chronic nicotine administration increased nNOS expression in the hippocampus
of female, but not male rats [16]. Exposure to stressors during
adolescence increases vulnerability to psychostimulant abuse, and
female rats stressed during puberty show enhanced locomotor sensitization to nicotine treatment [21]. Although the emphasis of the
current study is not to study the effect of single housing stress on
the nitrergic system in rat brain, we have to consider this effect
when discussing the nicotine-induced changes in the levels of NO
metabolites. Subsequently, the higher levels of NO metabolites in
rats exposed to nicotine as adolescents compared to those exposed
as adults may be reflecting the effect of pubertal stress exposure.
In the mammalian brain, nine of the total 12 nAChR subunit
genes are expressed: ␣2–␣7, ␤2–␤4. Of these, ␣4, ␤2 and ␣7 subunits are the most abundant and widespread in the rodent brain
(reviewed in [7,12,13]). Genetic analyses and manipulations point
to the involvement of receptors containing ␣4, ␤2 and ␣7 subunits
in nicotine addiction [2,8]. The frontal cortex, hippocampus and
amygdala are rich in these three types of subunits. Therefore, the
subunit composition of the nACh receptors is unlikely to underlie
the differences in NO activity in these three brain regions.
The present study can be considered an observational study; we
did not employ a mechanistic approach. We used an outbred rat
strain (Sprague Dawley) and preselected rats (minimum and maximum nicotine preferring) based on their nicotine consumption in a
two-bottle free choice oral nicotine intake paradigm. To study the
effect of age at onset of nicotine exposure, we used two groups of
rats: rats first exposed to nicotine as adolescents or as adults. Rats
exposed to the nicotine choice as adolescents were singly housed
for 13 weeks and received nicotine for 12 weeks, while rats exposed
as adults were housed singly for seven weeks and received nicotine
for six weeks. Our findings show that the amount of nicotine consumed does not affect the levels of nitric oxide metabolites in the
three brain regions studied. If the different nicotine preferences
and intake of the rats have a genetic basis, then our results suggest
that genes regulating NO activity and nicotine preference are not
related. On the other hand, there was a significant effect of age at
onset in the frontal cortex; adult-onset animals had lower levels
of NOx compared to adolescent-onset animals. This difference may
be attributed to the effect of longer isolation stress or to the effect
of extended nicotine intake. However, considering that isolation
stress affected NOx levels in the hippocampus but not in the frontal
105
cortex when naïve rats were compared to singly housed control
animals, the effect of nicotine is more likely to underlie this difference. Adolescence is a critical phase in development and adolescent
nicotine exposure produces long-lasting neurochemical effects that
persist into adulthood in rodents (reviewed in [15,31]). Our findings
suggest that the neuroplastic changes induced by nicotine exposure starting in adolescence resulted in increased NO activity in
adulthood.
There are a few reports on the comparison of NOS expression
in adult and adolescent brain regions. Terada et al. [33] studied the ontogeny of nNOS in the developing rat brain (between
embryonic day 13 and postnatal day 14). It is reported that nNOSpositive cells at postnatal day 14 resemble those in the adult rat
brain. Neuronal NOS mRNA is widely distributed in the rat brain,
including the olfactory bulbs, the islands of Calleja, the amygdala,
thalamus, hypothalamus, cerebellum, cerebral cortex, basal ganglia, hippocampus and the brain stem [14]. It would be illuminating
to study the effect of nicotine intake in rats preselected for nicotine
preference in brain regions rich in dopaminergic innervation.
Neurotoxic insults are reported to increase nitric oxide metabolites in the brain. For example, METH-induced dopaminergic
neurotoxicity is accompanied by increases in the number of nNOS
expressing cells [10] and in NO metabolites in brain regions rich in
dopaminergic cells [3]. Glutamate was the first reported trigger that
increased NO levels in the brain (reviewed in [24]). Nicotine stimulates dopaminergic neurons through alpha-7-containing nicotinic
ACh receptors on glutamatergic afferents in the ventral tegmental
area; activated NMDA receptors increase NO levels. Schilstrom et al.
[30] suggest that increased NO production may be involved in plastic changes which underlie nicotine addiction. Along these lines, the
time-course of nicotine exposure may be related to the extent of the
plastic changes. In the current study, while the amount of nicotine
consumed by rats did not have an effect on NOx, adolescent-onset
rats had higher levels in the frontal cortex than adult-onset animals.
These findings suggest that nicotine, possibly through dopaminergic and glutamateric mechanisms, increases NOx in the frontal
cortex of rat brain, and that the increase is related to the duration and developmental stage of exposure rather than the amount
of nicotine consumed.
In conclusion, we have confirmed that isolation stress increases
brain NOx in male and female Sprague Dawley rats in the hippocampus. Nicotine preference did not affect brain NO activity,
implying differences in the genetic regulation of the nicotinic
cholinergic and nitrergic systems in rat brain. On the other hand,
age at onset of nicotine exposure had a significant impact on NOx
in the frontal cortex, suggesting long-lasting effects of nicotine on
NO synthesis in the frontal cortex. These findings underscore the
importance of nicotine exposure during adolescence.
Acknowledgements
This study was supported by Ege University Research Fund Grant
2006/BAM/001 to LK. The authors would like to thank Jacqueline
Renee Gutenkunst, (Izmir University of Economics, School of Foreign Languages) for her effort in refining the language of this paper.
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