Download Effects of chronic nicotine administration on nitric oxide synthase

Journal of Neuroscience Research 67:689 – 697 (2002)
DOI 10.1002/jnr.10158
Effects of Chronic Nicotine Administration
on Nitric Oxide Synthase Expression and
Activity in Rat Brain
Eduardo Weruaga,1 Burcu Balkan,2 Ersin O. Koylu,2 Sakire Pogun,2 and
José R. Alonso1*
1
Department of Cell Biology and Pathology and Institute for Neuroscience of Castilla y León, University of
Salamanca, Salamanca, Spain
2
Ege University Center for Brain Research and School of Medicine, Department of Physiology and Basic
Neuroscience Research Unit of TUBITAK, Bornova, Izmir, Turkey
Although there is substantial evidence concerning the
influence of nicotine on nitric oxide (NO) synthesis in the
vascular system, there are fewer studies concerning the
central nervous system. Although NO metabolites
(nitrates/nitrites) increase in several rat brain regions after
chronic injection of nicotine, the cellular origin of this rise
in NO levels is not known. The aim of the present work
was to assess the effects of repetitive nicotine administration on nitric oxide synthase (NOS) expression and
activity in male and female rat brains. To determine levels
of nitrate/nitrite, the Griess reaction was carried out in
tissue micropunched from the frontal cortex, striatum,
and accumbens of both male and female rats untreated
(naı̈ve) or injected with saline or nicotine (0.4 mg/kg for 15
days). In parallel, coronal sections of fixed brains from
equally treated animals were immunostained for neuronal
NOS or histochemically labelled for NADPH-diaphorase
activity. Nicotine treatment increased NO metabolites
significantly in all brain regions compared with naı̈ve or
saline-treated rats. By contrast, analysis of the planimetric counting of NOS/NADPH-diaphorase-positive neurons failed to demonstrate any significant effect of the
nicotine treatment. A significant decrease was observed
with both techniques employed in saline-injected female
rats compared with naı̈ve animals, suggesting a stress
response. The mismatch between the biochemical and
the histological data after chronic nicotine treatment is
discussed. The up-regulation of NO sources other than
neurons is proposed. © 2002 Wiley-Liss, Inc.
Key words: addiction; drugs of abuse; NADPH-diaphorase; neurotransmitter modulation; sex differences
Nicotine exerts its central actions by regulating cationic fluxes through nicotinic acetylcholine receptors
(nAChR) on neurons that use acetylcholine, dopamine,
norepinephrine, serotonin, glutamate, or ␥-aminobutyric
acid (GABA) as transmitters (Kaiser and Wonnacott, 2000;
Wonnacott et al., 2000). nAChRs are present, both preand postsynaptically, in brain regions critical for cognitive
© 2002 Wiley-Liss, Inc.
function and addiction, namely, the cortex, striatum, and
ventral tegmental area (Levin, 1992; Levin and Simon,
1998). Although it has been well established that nAChR
activation can lead to the entry of Na⫹ into the cell, the
evidence also supports the notion that activation of some
nAChRs leads to enhanced entry of Ca2⫹ (Rosecrans and
Karan, 1993). Through this effect, the drug also probably
modifies events occurring beyond the nAChR, including
the regulation of nitric oxide (NO) synthesis.
For the nervous system, assessment of the interactions between nicotine and NO synthesis was first reported in the periphery. In the gastrointestinal system,
nicotine liberates NO from nonadrenergic noncholinergic nerves involved in the relaxation of the sphincter
of Oddi, where neurons containing NADPHdiaphorase (NO-synthesizing cells) have been demonstrated (Tanobe et al., 1995). In rats chronically exposed
to cigarette smoke, neither erectile responses nor endothelial nitric oxide synthase (NOS) protein levels are
affected, but NOS enzymatic activity—and hence NO
production—is decreased; on the other hand, nicotine
substantially decreases the neuronal isoform of NOS
(nNOS; Xie et al., 1997).
For the central nervous system, NO-nicotine interactions have also been reported. Stevens et al. (1997)
showed that nicotine might modulate inhibitory gating
Contract grant sponsor: NATO; Contract grant number: CRG-972168;
Contract grant sponsor: TUBITAK; Contract grant number: SBAG-U/151.3; Contract grant sponsor: Ege University Research Fund; Contract grant
number: 98-TIP-009; Contract grant sponsor: Junta de Castilla y León,
DGES (PM99-0068); Contract grant number: FEDER-CICYT (1FD971325); Contract grant sponsor: Ministerio del Interior, Plan Nacional Sobre
Drogas.
*Correspondence to: Dr. José R. Alonso, Depto. Biologı́a Celular y Patologı́a, Facultad de Medicina, Universidad de Salamanca, Avda. Alfonso X
el Sabio 1, E-37007 Salamanca, Spain. E-mail: [email protected]
Received 27 March 2001; Revised 6 October 2001; Accepted 1 November
2001
Published online 31 January 2002
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Weruaga et al.
in rat hippocampus through NO, where nAChRs
(bungarotoxin-binding sites) have been well documented
in NOS-containing neurons (Adams and Stevens, 1998).
In fact, Adams et al. (2000) have shown that the hippocampal auditory gating is indeed mediated through NO
release, through activation of the ␣7 subtype of nAChR.
There are few studies of NO mediation as regards the
biobehavioral effects of nicotine. Suemaru et al. (1997)
have shown that chronic nicotine treatment induces tail
tremor in rats, which is attenuated by prior NOS inhibition or MK-801 application, suggesting the involvement
of NO formation through N-methyl-D-aspartate
(NMDA) receptors in this behavioral response to nicotine.
Levin et al. (1998) have demonstrated the attenuation by
nicotine of the impairment in working memory performance caused by MK-801 in rats. Yilmaz et al. (2000)
have demonstrated the blockage of the nicotine-induced
enhancement of active avoidance learning in rats by prior
NOS inhibition.
Recently, Pögün et al. (2000) have demonstrated
that acute and chronic nicotine administration (0.4 mg/kg,
sc, once or for 15 days, respectively) to rats increases the
levels of NO2–/NO3–, stable metabolites of NO, in several brain regions. Differences were seen in the nicotine
response with regard to sex, degree of stimulation, brain
regions affected, and duration of treatment (i.e., acute vs.
chronic). However, prior inhibition of NOS eliminated
the effect of nicotine in all regions studied.
NADPH-diaphorase (ND) histochemical staining
is a selective marker for distinct neural populations
widely distributed throughout the central nervous system (Vincent and Kimura, 1992; Alonso et al., 2000).
ND-activity may be localized in both fixed and unfixed
brain tissue (Thomas and Pearse, 1961; Alonso et al.,
1995). Different approaches have revealed that the NDstaining corresponds to NOS expression (Dawson et al.,
1991; Hope et al., 1991; Snyder, 1995); hence, both
methods can be used to study the nitrergic system.
Although ND activity and nNOS (NOS I; EC
1.14.13.39) immunoreactivity also colocalize in astrocytes (Kugler and Drenckhahn, 1996), this labelling can
be eliminated by appropriate fixation with paraformaldehyde (Kishimoto et al., 1993; Gabbott and Bacon,
1996). Hence, under defined methodological conditions, ND histochemistry is an easy method to detect
neurons producing the neuroactive molecule NO. Furthermore, some evidence suggests that ND histochemical activity would be directly related to NO synthesis
(Morris et al., 1997; Weruaga et al., 2000).
The present study was undertaken to assess the effects
of chronic nicotine administration on NOS expression and
activity in male and female rat brains. Biochemical determination of the stable metabolites of NO was carried out
in parallel with histochemical and immunohistochemical
determination of neuronal NOS-positive neurons in brain
regions involved in the addictive and cognition-enhancing
properties of nicotine.
MATERIALS AND METHODS
Experimental Animals
Sexually mature male and female rats (Rattus norvegicus,
Sprague-Dawley, 3 months of age, 220 –250 g) were used in the
experiments. The rats were kept under standard conditions
(three or four per cage, 20 –22°C, 12 hr light/dark cycle) with
food and water provided ad libitum. The animals were handled
at all times under the prescriptions for animal care and experimentation of the pertinent European Communities Council
Directive (86/609/EEC), current Spanish legislation (BOE 67/
8509-12, 1988), and the Institutional Animal Ethics Committee
of Ege University.
Six experimental groups were used for the histological and
biochemical analyses: naı̈ve, saline-, or nicotine-treated males
and females. Each group included 12 animals, five for the
histological analyses and seven for the biochemical assays. Thus,
the total number of rats used was 72.
Drugs
(–)-Nicotine hydrogen tartrate (Sigma, St. Louis, MO;
N-5260) was dissolved in isotonic saline (0.9% w/v NaCl), and
the pH of the solution was adjusted to 7.0 with diluted NaOH.
The nicotine dose (0.4 mg/kg body weight) was calculated as
the base and administered subcutaneously once daily at 8:00 –
9:00 hr over 15 days. Control animals received saline under the
same regimen. All animals were sacrificed 24 hr after the last
injection.
Histological Preparations
Rats destined for histological analyses were deeply anesthetized with sodium pentobarbital (Nembutal; 40 mg/kg, ip)
and sacrificed by intracardiac perfusion. Initially, isotonic saline
was used to flush out the blood, after which a mixture containing 4% (w/v) depolymerized paraformaldehyde and 0.2% (w/v)
picric acid in 0.1 M phosphate buffer (PB), pH 7.3, was perfused
for 15 min. Brains were dissected out, cut into blocks, and
postfixed in the same solution for 4 hr at 4°C. Tissue blocks
were cryoprotected with 30% (w/v) sucrose in PB until they
sank, after which they were quickly frozen and stored at – 80°C.
Tissue blocks were cut at 25 ␮m along coronal planes and
collected in six series in cold PB. From each brain block, a
one-in-six series was Nissl stained, another was histochemically
stained for ND activity, and a third was stained immunohistochemically for neuronal NOS (nNOS). Some series were doubly processed for both immuno- and histochemical techniques
as controls. The remaining series were used for other purposes.
Nissl Staining
Free-floating sections were postfixed for 1 week in standard buffered formalin. After being mounted onto gelatincoated slides, they were dehydrated in an increasing ethanol
series, followed by a mixture of chloroform:ethanol (1:1). Next,
they were stained with 0.25% (w/v) thionine (Merck, Darmstadt, Germany) and dehydrated again, cleared with xylene, and
coverslipped with Entellan (Merck).
ND Histochemistry
Sections were incubated at 37°C in the darkness in a
medium containing 0.08 % (w/v) Triton X-100 (Riedel de
Nicotine and Nitric Oxide Synthase
Haen; 56029), 0.8 mM nitroblue tetrazolium (Sigma; N-6876),
and 1 mM ␤-NADPH (Sigma; N-7505) in 0.1 M Tris-HCl, pH
8.0, for 60 min, the reaction being controlled under the microscope. To ensure reliable comparisons among the different
groups, one series from one animal from each group (i.e., six
series of sections from naı̈ve, nicotine-, or saline-treated male
and female rats) was included in a parallel staining protocol using
the same incubation medium (see Evaluation below). Controls
for specificity of the ND histochemistry were carried out as
previously described (Alonso et al., 1995). No reaction product
was observed in the tissue when incubated without NADPH or
without chromogen. Stained sections were mounted onto
gelatin-coated slides, dried overnight at 37°C, dehydrated,
cleared with xylene, and coverslipped with Entellan.
Neuronal NOS Immunohistochemistry
The sections were incubated at 4°C for 3 days in a
medium containing 1:15,000 anti-nNOS sheep IgG (Dr. Emson
and Charles, Cambridge, United Kingdom), 1% (v/v) normal
rabbit serum (Vector Laboratories, Burlingame, CA), and 0.05%
(w/v) Triton X-100 in PB. After thorough washing with PB
(5 ⫻ 10 min), the sections were incubated at room temperature
with biotinylated anti-sheep rabbit IgG (1:200; Vector) and 1%
normal rabbit serum in PB for 60 min. They were then rinsed
again in PB (5 ⫻ 10 min) and, finally, incubated with avidinbiotin complex (1:250; Vectastain ABC Kit; Vector) for 90 min
at room temperature. Tissue-bound peroxidase was revealed by
incubating the sections with 0.003% (v/v) H2O2 and 0.02%
(w/v) 3,3⬘-diaminobenzidine (DAB; Sigma; D-5637) in 0.05 M
Tris-HCl, pH 7.6, the reaction being controlled under the
microscope. The whole procedure was applied equally for slices
coming from animals from the six different groups as for ND
staining. Sections were mounted on slides as described above.
Nitrite and Nitrate Determination
Rats destined for biochemical analysis were decapitated,
and their brains were rapidly removed, placed on ice, and sliced
(1 mm thick). Selected brain punches were obtained from the
regions depicted in Figure 1, at the same levels where histological analyses were performed. Tissue was wet weighed and
frozen at – 40°C.
Nitrite and nitrate levels were determined in tissue samples as described previously, with slight modifications (Taskiran
et al., 1997). Samples were homogenized in PB, pH 7.5, and
centrifuged at 2,000g for 5 min at 4°C. The supernatants were
incubated 2:1 with 0.3 M NaOH for 5 min and thereafter 3:1
with 5% (w/v) ZnSO4 for deproteinization. The mixture was
centrifuged again at 3,000g for 20 min, and the supernatants
were used for NO3–/NO2– determinations. Preexisting nitrite
levels were directly measured by the Griess reaction (Green et
al., 1982), whereas nitrates were first reduced by nitrate reductase (EC 1.6.6.2.) from Aspergillus (Boehringer Mannheim) in
the presence of NADPH and FAD (Bories and Bories, 1995).
Total nitrites (reduced NO3– and preexisting NO2–) were evaluated spectrophotometrically at 546 nm and considered as total
stable metabolites of NO. Sodium nitrite and nitrate solutions
(Merck) were used for preparing the standard solutions. The
results are expressed in units of ␮mol/g wet weight of tissue.
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Evaluation
Histochemistry and immunohistochemistry. The
boundaries of the different regions were determined, and NDstained cells were counted by the same author (B.B.). These
planimetric analyses were performed in three consecutive sections from the same series (150 ␮m apart), corresponding to
Bregma levels ranging from 1.20 to 1.70 mm (Paxinos and
Watson, 1998). Rectangular frames (200 ⫻ 215 ␮m) were
captured with a 20⫻ objective and a color video camera (JVC
TK-1280E), and positive neurons were counted semiautomatically in each frame. ND-positive neurons were counted only
when second-order dendrites were distinguished. In total 81
frames from cortex (in three well-defined axes), 48 from corpus
striatum (caudate putamen nucleus, avoiding edges with external
capsule), and 37 from accumbens nucleus (rostral, central, and
caudal parts) were analyzed for each animal. This procedure
allowed us to compare the total numbers of ND/NOS-positive
cells within the designated regions among experimental groups,
because similar sectional areas were analyzed in each animal.
Because staining was performed in groups of six animals, as
described above, the percentage of cells for each saline- or
nicotine-treated animal was calculated relative to that of naı̈ve
animals of the same sex, and the statistical evaluation was carried
out accordingly: ANOVAs were performed to determine the
variance, with sex (male, female) and treatment (saline, nicotine)
as factors.
Nitrite and nitrate determinations. In the statistical
analysis of the biochemical determinations, ANOVAs were employed, with sex (male, female), treatment (naı̈ve, saline, nicotine), and brain regions (cortex, striatum, accumbens nucleus) as
the factors and NO3–/NO2– levels as the dependent variable.
Duncan’s post hoc analyses were performed as required. To
allow for comparisons between the results obtained in the biochemical and histological analyses, the results from both types of
experiment are represented as percentages of the naı̈ve animals’
values for each saline- or nicotine-treated group.
RESULTS
Histology
Neuronal NOS immunoreactivity and ND activity
colocalized in the same population of neurons in the three
regions were studied after double-labelling analyses (not
shown) and comparing both types of staining (Fig. 1).
However, the stain obtained with the histochemical technique provided more details about the morphology of the
positive elements than the immunohistochemical procedure, i.e., a higher degree of branching. Therefore, we
employed ND staining for quantitative analyses. Positive
neurons in the three regions studied were seen to correspond to type I ND elements and mainly had multipolar
branching dendrites (Fig. 1).
Because the experiments were carried out in groups
of six animals, one animal from each group, data evaluation was carried out accordingly. The ND/NOS-positive
neuron counts obtained from each brain region of
nicotine- or saline-treated rats were represented as the
percentages of the counts for naı̈ve animals for each sex
separately. Figure 2 depicts the percentage difference from
Fig. 1. Adjacent coronal sections from a male naı̈ve rat immunolabelled
for neuronal NOS (nNOS; A,C–E) or stained with the histochemical
reaction for NADPH-diaphorase (ND; B,F–H). A,B: Panoramic views
of the central rostrocaudal level where quantitative biochemical and
histochemical analyses were performed. Details from the three regions
studied, frontal cortex (Cx; C,F), dorsal striatum (caudate putamen, Str;
D,G), and ventral striatum (accumbens nucleus, Acb; E,H) are also
depicted. NADPH-diaphorase staining resulted in labelling of blood
vessels, which was more patent in the cerebral cortex (see B,F). However, at higher magnification, ND-stained neural elements (F–H) are
more easily distinguishable compared with immunolabelling (C–E).
Both types of techniques employed yield the staining of similar neuronal populations, with similar cell densities in each region. Scale
bars ⫽ 1 mm for A,B, 50 ␮m for C–H.
Nicotine and Nitric Oxide Synthase
Fig. 2. Percentage difference of the number of ND/NOS-positive
neurons in nicotine- or saline-treated rats compared with naı̈ve animals
in the three regions studied in male and female rats. Bars represent
means ⫾ SEM. The asterisk indicates a statistically significant difference
from naı̈ve rats, same sex, same brain region (Duncan’s post hoc t-test,
P ⫽ 0.017).
naı̈ve male and female rats in groups that received saline or
nicotine injections. Multifactorial ANOVA revealed a significant main effect of treatment [F(2,89) ⫽ 3.29; P ⫽
0.043], implying a difference among naı̈ve, saline-, and
nicotine-treated rats with regard to labelled cells numbers.
When post hoc tests were applied separately for groups,
the only significant difference (P ⫽ 0.017) was observed in
the corpus striatum of female rats, where saline injections
lowered the number of ND/NOS-positive neurons (85%
of naı̈ve). Figure 3 shows the corpus striatum of females
from the three experimental groups. Table I gives both
number and cell densities in the three brain regions studied.
Biochemistry
A multifactorial ANOVA with sex (male, female),
treatment (naı̈ve, saline, or nicotine), and brain region
(cortex, striatum, accumbens) as the factors and NO2–/
NO3– levels as the dependent variable revealed significant
693
main effects of brain regions [F(2,102) ⫽ 7.36; P ⬍ 0.001]
and treatment [F(2,102) ⫽ 43.18; P ⬍ 0.0001] and an
interaction between brain regions and treatment
[F(4,102) ⫽ 6.21; P ⬍ 0.0001]. As depicted in Figure 4
nicotine caused a significant increase in NO2–/NO3– levels in all brain regions in both sexes compared with the
naı̈ve or saline-treated animals. Post hoc analyses, in
addition to showing that nicotine caused a significant
increase in all regions tested, also revealed that saline
injections were not inert and lowered cortical NO2–/
NO3– levels in female rats: The saline-treated females had
significantly lower cortical NO2–/NO3– levels than naı̈ve
females.
Figure 5 shows the percentage difference in naı̈ve
male and female rats in the groups that received saline or
nicotine injections. Comparison of Figure 5 (biochemistry) with Figure 2 (histochemistry) reveals that the response to nicotine as measured by changes in brain NO2–/
NO3– levels is substantially higher than that measured by
nNOS histochemical expression.
DISCUSSION
In the present work an increase in the production of
NO in three different brain regions (frontal cortex, corpus
striatum, and accumbens nucleus) was seen after repetitive
experimental administration of nicotine (chronic treatment), as reflected by an increase in the levels of nitrates
and nitrites. Moreover, this is the first time a comparison
is offered concerning the effect of nicotine on NO production using both biochemical and histological approaches, in that the ND histochemical expression was
analyzed in neurons of the same brain regions in parallel.
The histochemical and immunohistochemical studies of
ND/NOS-positive neurons also revealed a similar trend;
however, the magnitude of the response was not nearly as
high as that reflected in NO2–/NO3– levels. The results of
the present study confirm the observations of a previous
study by Pögün et al. (2000) and show that chronic
nicotine treatment in rats increases the levels of stable
metabolites of NO in both male and female brains.
One aspect of this study was the demonstration of
diminished NOS expression because of the stress induced
by chronic injections. There was a parallel variation in NO
metabolites. In both measurements, this effect reached
significant levels in female rats. This finding supports the
view that NO may be involved in the stress response (for
review see López-Figueroa et al., 1998). In studies carried
out under diverse stressors employing biochemical methods or the ND histochemical technique (Kishimoto et al.,
1996; Vaid et al., 1997; Sánchez et al., 1999; Veenman et
al., 1999), increases in the NOS activity and/or expression
have been reported in the limbic-hypothalamic-pituitaryadrenal axis. Thus, we describe for the first time a sexually
dimorphic change in NOS expression and activity in
response to chronic injections out of the stress axis (i.e.,
the striatum and cerebral cortex).
The effects of nicotine on the nitrergic system were
not the same in both sexes. The results obtained are in
agreement with other animal studies in which male and
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Weruaga et al.
female rodents were found to show different sensitivities
to the effects of nicotine. For example, food intake and
physical activity (Bowen et al., 1986; Grünberg et al.,
1986), suppression of Y-maze activity (Hatchell and Col-
lins, 1980), active avoidance learning (Yilmaz et al., 1997),
up-regulation of [3H]cystine binding sites (Koylu et al.,
1997), induced antinociception (Craft and Milholland,
1998; Chiari et al., 1999; Lavand’homme and Eisenach,
1999; Damaj, 2001), prepulse inhibition (Faraday et al.,
1999), and locomotor activity (Booze et al., 1999; Kanit et
al., 1999) display sexual dimorphism when progressing
under the influence of nicotine. Despite the huge differences in the experimental animal used; the dose, route,
and duration of the drug administration; and the biobehavioral task employed, the evidence is that nicotine exerts
most of its influence on brain physiology following a
sexually dimorphic pattern. The mechanisms underlying
this sex difference in nicotine effects, including those
related to brain NO production, remain uncertain, but
recent in vivo and in vitro evidence strongly suggests that
sex hormones can modulate the activation of the ␣4␤2
neuronal acetylcholine receptors and the blockage of antinociception by nicotine (Damaj, 2001).
Our results indicate significant differences in the
response to chronic treatment with nicotine on measuring
NO metabolites levels and the number of neurons positive
for NOS/ND. There are at least three possible explanations for this divergence. First, the immunohistochemical
and histochemical localization of NOS/ND within each
positive cell is not a quantitative indicator of NO production: With these methods the presence of the enzyme or
its activity is detected, but they fail to demonstrate how
much NO is actually produced. It is therefore possible that
there was indeed an increase in the NO stable metabolites
after nicotine injection (produced by the same NOS/NDpositive neurons) but no de novo expression of the enzyme, the number of counted positive neurons thus increasing. The degree of staining of ND-positive neurons
may be related to the level of NOS enzyme activation
(DellaCorte et al., 1995; Morris et al., 1997; Weruaga et
al., 2000), but the former histological quantification is not
reliable enough to ensure that the positive neurons
counted produce more neurotransmitter. Second, the mismatch between the biochemical and the histochemical
results may be due to production of NO from sources
other than neurons. Nonactivated astrocytes are an important site of nNOS, but its histological demonstration must
be made either with special tissue fixation and freezing or
with weak formaldehyde fixation (Gabbott and Bacon,
1996; Kugler and Drenckhahn, 1996), which is, on the
other hand, incompatible with both the discrimination of
neuronal elements and the quantification of their density
in large brain regions. ND staining related to inducible
NOS (NOS II) can be visualized with “standard” fixation,
but it is seen only after being up-regulated in activated
Fig. 3. The three panels correspond to the striatum (caudate putamen)
of naı̈ve (A), saline-injected (B), and nicotine-injected (C) female rats
on sections stained histochemically for NADPH-diaphorase. Quantitative analyses of stained cells densities demonstrated a statistically
significant decrease only in the saline group compared with the naı̈ve
(untreated) or the nicotine-injected animals. Scale bar ⫽ 100 ␮m.
Nicotine and Nitric Oxide Synthase
695
TABLE I. Number and Density of NOS/ND-Positive Cells*
Cortex
Male
Naı̈ve
Control
Nicotine
Female
Naı̈ve
Control
Nicotine
Striatum
Accumbens
Cell count
Density
Cell count
Density
Cell count
Density
27.4 ⫾ 3.2
28.6 ⫾ 5.1
33.4 ⫾ 3.7
17.2 ⫾ 2.0
18.0 ⫾ 3.2
21.0 ⫾ 2.4
103.4 ⫾ 9.9
91.0 ⫾ 6.9
96.6 ⫾ 4.7
65.0 ⫾ 6.2
57.2 ⫾ 4.4
60.7 ⫾ 3.0
71.0 ⫾ 3.5
69.4 ⫾ 7.9
78.8 ⫾ 2.3
44.6 ⫾ 2.2
43.6 ⫾ 4.9
50.0 ⫾ 1.4
29.5 ⫾ 2.9
25.6 ⫾ 2.6
26.4 ⫾ 3.6
18.5 ⫾ 1.8
16.1 ⫾ 1.6
16.6 ⫾ 2.3
102.8 ⫾ 6.5
87.2 ⫾ 5.0
98.6 ⫾ 6.9
64.6 ⫾ 4.1
54.8 ⫾ 3.2
62.0 ⫾ 4.3
70.2 ⫾ 7.3
59.8 ⫾ 4.9
70.2 ⫾ 5.7
44.1 ⫾ 4.6
37.6 ⫾ 3.2
44.1 ⫾ 3.6
*Cell count: The average number of total cells counted (mean ⫾ SEM); density: The average cell density/mm2 (mean ⫾ SEM).
Fig. 4. Total measurements of NO2–/NO3– levels in nicotine- or
saline-treated rats compared with naı̈ve animals. Bars are grouped by
brain regions and represent means ⫾ SEM. Symbols on the bars
represent different degrees of statistical significance for differences between naı̈ve animals and nicotine-injected rats (*P ⬍ 0.05, **P ⬍ 0.01,
***P ⬍ 0.0005) or saline-injected rats (#P ⬍ 0.05) of the same sex and
in the same brain region, as post hoc Duncan’s analyses.
astrocytes or microglial cells in diverse brain insults (Lei et
al., 1996; Wallace, 1996; De Groot et al., 1997; Ding et
al., 1997; Wallace et al., 1997; Stojkovic et al., 1998).
However, to our knowledge, there is no documentation
of enhanced NO production from glial cells resulting from
nicotine treatment. Because nicotinic receptors are functional in astrocytes (Hosli et al., 1997, 2000), studies are
currently being undertaken to clarify this issue.
Finally, another source of NO is blood vessels, which
contain endothelial NOS (eNOS; NOS III; for review see
Moncada, 1997). As shown in Figure 1, ND histochemical
staining labels blood vessels. This staining corresponds to
eNOS rather than nNOS. However, the histological
quantification of these is probably not as feasible as
neuronal staining. Recent evidence has been found
supporting this possibility: Using the L-[3H]arginine and
3
L-[ H]citrulline assay and different enzyme preparations,
Tonnessen et al. (2000) have demonstrated that nicotine
increases the activity of eNOS more than that of nNOS
and has no effect on inducible NOS. Thus, it is more
likely that nicotine would enhance NO2–/NO3– levels
mainly through the eNOS located in blood vessels rather
than through the neuronal isoform detected by histolog-
Fig. 5. Percentage difference of NO2–/NO3– levels in nicotine- or
saline-treated rats compared with naı̈ve animals in the three regions
studied in male and female rats. Bars represent means ⫾ SEM. For post
hoc statistical evaluation, see Figure 4.
ical techniques. Pharmacological assays using selective
NOS isoform inhibitors could be used to test this hypothesis.
It may be concluded that, although nicotine causes
an overall increase in NOS positive neurons, the stress
arising from chronic injections reduces the number of
NOS-positive neurons and that this latter effect is most
prominent in the female corpus striatum. Biochemical
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Weruaga et al.
assays of NO2–/NO3– in rat brain following nicotine
treatment reveal a greater level of NO production compared with the number of NOS/ND-positive neurons,
suggesting the involvement of eNOS and/or other nonneuronal sources of nNOS.
ACKNOWLEDGMENTS
The authors thank Dr. Dilek Taskiran for her guidance in the biochemical assays. The help of N. Skinner in
revising the English version of the manuscript is acknowledged.
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