Effects of Chronic Nicotine Administrationon Nitric Oxide Synthase Expression andActivity in Rat Brain

Eduardo Weruaga,1 Burcu Balkan,2 Ersin O. Koylu,2 Sakire Pogun,2 andJose R. Alonso1*1Department of Cell Biology and Pathology and Institute for Neuroscience of Castilla y Leon, University ofSalamanca, Salamanca, Spain2Ege University Center for Brain Research and School of Medicine, Department of Physiology and BasicNeuroscience Research Unit of TUBITAK, Bornova, Izmir, Turkey

Although there is substantial evidence concerning theinfluence of nicotine on nitric oxide (NO) synthesis in thevascular system, there are fewer studies concerning thecentral nervous system. Although NO metabolites(nitrates/nitrites) increase in several rat brain regions afterchronic injection of nicotine, the cellular origin of this risein NO levels is not known. The aim of the present workwas to assess the effects of repetitive nicotine adminis-tration on nitric oxide synthase (NOS) expression andactivity in male and female rat brains. To determine levelsof nitrate/nitrite, the Griess reaction was carried out intissue micropunched from the frontal cortex, striatum,and accumbens of both male and female rats untreated(naıve) or injected with saline or nicotine (0.4 mg/kg for 15days). In parallel, coronal sections of fixed brains fromequally treated animals were immunostained for neuronalNOS or histochemically labelled for NADPH-diaphoraseactivity. Nicotine treatment increased NO metabolitessignificantly in all brain regions compared with naıve orsaline-treated rats. By contrast, analysis of the planimet-ric counting of NOS/NADPH-diaphorase-positive neu-rons failed to demonstrate any significant effect of thenicotine treatment. A significant decrease was observedwith both techniques employed in saline-injected femalerats compared with naıve animals, suggesting a stressresponse. The mismatch between the biochemical andthe histological data after chronic nicotine treatment isdiscussed. The up-regulation of NO sources other thanneurons is proposed. © 2002 Wiley-Liss, Inc.

Key words: addiction; drugs of abuse; NADPH-diapho-rase; neurotransmitter modulation; sex differences

Nicotine exerts its central actions by regulating cat-ionic fluxes through nicotinic acetylcholine receptors(nAChR) on neurons that use acetylcholine, dopamine,norepinephrine, serotonin, glutamate, or �-aminobutyricacid (GABA) as transmitters (Kaiser and Wonnacott, 2000;Wonnacott et al., 2000). nAChRs are present, both pre-and postsynaptically, in brain regions critical for cognitive

function and addiction, namely, the cortex, striatum, andventral tegmental area (Levin, 1992; Levin and Simon,1998). Although it has been well established that nAChRactivation can lead to the entry of Na� into the cell, theevidence also supports the notion that activation of somenAChRs leads to enhanced entry of Ca2� (Rosecrans andKaran, 1993). Through this effect, the drug also probablymodifies events occurring beyond the nAChR, includingthe regulation of nitric oxide (NO) synthesis.

For the nervous system, assessment of the interac-tions between nicotine and NO synthesis was first re-ported in the periphery. In the gastrointestinal system,nicotine liberates NO from nonadrenergic noncholin-ergic nerves involved in the relaxation of the sphincterof Oddi, where neurons containing NADPH-diaphorase (NO-synthesizing cells) have been demon-strated (Tanobe et al., 1995). In rats chronically exposedto cigarette smoke, neither erectile responses nor en-dothelial nitric oxide synthase (NOS) protein levels areaffected, but NOS enzymatic activity—and hence NOproduction—is decreased; on the other hand, nicotinesubstantially decreases the neuronal isoform of NOS(nNOS; Xie et al., 1997).

For the central nervous system, NO-nicotine inter-actions 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/15-1.3; Contract grant sponsor: Ege University Research Fund; Contract grantnumber: 98-TIP-009; Contract grant sponsor: Junta de Castilla y Leon,DGES (PM99-0068); Contract grant number: FEDER-CICYT (1FD97-1325); Contract grant sponsor: Ministerio del Interior, Plan Nacional SobreDrogas.

*Correspondence to: Dr. Jose R. Alonso, Depto. Biologıa Celular y Pa-tologıa, Facultad de Medicina, Universidad de Salamanca, Avda. Alfonso Xel Sabio 1, E-37007 Salamanca, Spain. E-mail: jralonso@usal.es

Received 27 March 2001; Revised 6 October 2001; Accepted 1 November2001

Published online 31 January 2002

Journal of Neuroscience Research 67:689–697 (2002)DOI 10.1002/jnr.10158

© 2002 Wiley-Liss, Inc.

in rat hippocampus through NO, where nAChRs(bungarotoxin-binding sites) have been well documentedin NOS-containing neurons (Adams and Stevens, 1998).In fact, Adams et al. (2000) have shown that the hip-pocampal auditory gating is indeed mediated through NOrelease, through activation of the �7 subtype of nAChR.

There are few studies of NO mediation as regards thebiobehavioral effects of nicotine. Suemaru et al. (1997)have shown that chronic nicotine treatment induces tailtremor in rats, which is attenuated by prior NOS inhibi-tion or MK-801 application, suggesting the involvementof NO formation through N-methyl-D-aspartate(NMDA) receptors in this behavioral response to nicotine.Levin et al. (1998) have demonstrated the attenuation bynicotine of the impairment in working memory perfor-mance caused by MK-801 in rats. Yilmaz et al. (2000)have demonstrated the blockage of the nicotine-inducedenhancement of active avoidance learning in rats by priorNOS inhibition.

Recently, Pogun et al. (2000) have demonstratedthat acute and chronic nicotine administration (0.4 mg/kg,sc, once or for 15 days, respectively) to rats increases thelevels of NO2

–/NO3–, stable metabolites of NO, in sev-

eral brain regions. Differences were seen in the nicotineresponse with regard to sex, degree of stimulation, brainregions affected, and duration of treatment (i.e., acute vs.chronic). However, prior inhibition of NOS eliminatedthe effect of nicotine in all regions studied.

NADPH-diaphorase (ND) histochemical stainingis a selective marker for distinct neural populationswidely distributed throughout the central nervous sys-tem (Vincent and Kimura, 1992; Alonso et al., 2000).ND-activity may be localized in both fixed and unfixedbrain tissue (Thomas and Pearse, 1961; Alonso et al.,1995). Different approaches have revealed that the ND-staining corresponds to NOS expression (Dawson et al.,1991; Hope et al., 1991; Snyder, 1995); hence, bothmethods can be used to study the nitrergic system.Although ND activity and nNOS (NOS I; EC1.14.13.39) immunoreactivity also colocalize in astro-cytes (Kugler and Drenckhahn, 1996), this labelling canbe eliminated by appropriate fixation with paraformal-dehyde (Kishimoto et al., 1993; Gabbott and Bacon,1996). Hence, under defined methodological condi-tions, ND histochemistry is an easy method to detectneurons producing the neuroactive molecule NO. Fur-thermore, some evidence suggests that ND histochem-ical activity would be directly related to NO synthesis(Morris et al., 1997; Weruaga et al., 2000).

The present study was undertaken to assess the effectsof chronic nicotine administration on NOS expression andactivity in male and female rat brains. Biochemical deter-mination of the stable metabolites of NO was carried outin parallel with histochemical and immunohistochemicaldetermination of neuronal NOS-positive neurons in brainregions involved in the addictive and cognition-enhancingproperties of nicotine.

MATERIALS AND METHODSExperimental Animals

Sexually mature male and female rats (Rattus norvegicus,Sprague-Dawley, 3 months of age, 220–250 g) were used in theexperiments. The rats were kept under standard conditions(three or four per cage, 20–22°C, 12 hr light/dark cycle) withfood and water provided ad libitum. The animals were handledat all times under the prescriptions for animal care and experi-mentation of the pertinent European Communities CouncilDirective (86/609/EEC), current Spanish legislation (BOE 67/8509-12, 1988), and the Institutional Animal Ethics Committeeof Ege University.

Six experimental groups were used for the histological andbiochemical analyses: naıve, saline-, or nicotine-treated malesand females. Each group included 12 animals, five for thehistological 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), andthe pH of the solution was adjusted to 7.0 with diluted NaOH.The nicotine dose (0.4 mg/kg body weight) was calculated asthe base and administered subcutaneously once daily at 8:00–9:00 hr over 15 days. Control animals received saline under thesame regimen. All animals were sacrificed 24 hr after the lastinjection.

Histological Preparations

Rats destined for histological analyses were deeply anes-thetized with sodium pentobarbital (Nembutal; 40 mg/kg, ip)and sacrificed by intracardiac perfusion. Initially, isotonic salinewas used to flush out the blood, after which a mixture contain-ing 4% (w/v) depolymerized paraformaldehyde and 0.2% (w/v)picric acid in 0.1 M phosphate buffer (PB), pH 7.3, was perfusedfor 15 min. Brains were dissected out, cut into blocks, andpostfixed in the same solution for 4 hr at 4°C. Tissue blockswere cryoprotected with 30% (w/v) sucrose in PB until theysank, after which they were quickly frozen and stored at –80°C.Tissue blocks were cut at 25 �m along coronal planes andcollected in six series in cold PB. From each brain block, aone-in-six series was Nissl stained, another was histochemicallystained for ND activity, and a third was stained immunohisto-chemically for neuronal NOS (nNOS). Some series were dou-bly processed for both immuno- and histochemical techniquesas controls. The remaining series were used for other purposes.

Nissl Staining

Free-floating sections were postfixed for 1 week in stan-dard buffered formalin. After being mounted onto gelatin-coated slides, they were dehydrated in an increasing ethanolseries, followed by a mixture of chloroform:ethanol (1:1). Next,they were stained with 0.25% (w/v) thionine (Merck, Darm-stadt, Germany) and dehydrated again, cleared with xylene, andcoverslipped with Entellan (Merck).

ND Histochemistry

Sections were incubated at 37°C in the darkness in amedium containing 0.08 % (w/v) Triton X-100 (Riedel de

690 Weruaga et al.

Haen; 56029), 0.8 mM nitroblue tetrazolium (Sigma; N-6876),and 1 mM �-NADPH (Sigma; N-7505) in 0.1 M Tris-HCl, pH8.0, for 60 min, the reaction being controlled under the micro-scope. To ensure reliable comparisons among the differentgroups, one series from one animal from each group (i.e., sixseries of sections from naıve, nicotine-, or saline-treated maleand female rats) was included in a parallel staining protocol usingthe same incubation medium (see Evaluation below). Controlsfor specificity of the ND histochemistry were carried out aspreviously described (Alonso et al., 1995). No reaction productwas observed in the tissue when incubated without NADPH orwithout chromogen. Stained sections were mounted ontogelatin-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 amedium containing 1:15,000 anti-nNOS sheep IgG (Dr. Emsonand Charles, Cambridge, United Kingdom), 1% (v/v) normalrabbit 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 temperaturewith biotinylated anti-sheep rabbit IgG (1:200; Vector) and 1%normal rabbit serum in PB for 60 min. They were then rinsedagain in PB (5 � 10 min) and, finally, incubated with avidin-biotin complex (1:250; Vectastain ABC Kit; Vector) for 90 minat room temperature. Tissue-bound peroxidase was revealed byincubating the sections with 0.003% (v/v) H2O2 and 0.02%(w/v) 3,3�-diaminobenzidine (DAB; Sigma; D-5637) in 0.05 MTris-HCl, pH 7.6, the reaction being controlled under themicroscope. The whole procedure was applied equally for slicescoming from animals from the six different groups as for NDstaining. 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 theregions depicted in Figure 1, at the same levels where histolog-ical analyses were performed. Tissue was wet weighed andfrozen at –40°C.

Nitrite and nitrate levels were determined in tissue sam-ples as described previously, with slight modifications (Taskiranet al., 1997). Samples were homogenized in PB, pH 7.5, andcentrifuged at 2,000g for 5 min at 4°C. The supernatants wereincubated 2:1 with 0.3 M NaOH for 5 min and thereafter 3:1with 5% (w/v) ZnSO4 for deproteinization. The mixture wascentrifuged again at 3,000g for 20 min, and the supernatantswere used for NO3

–/NO2– determinations. Preexisting nitrite

levels were directly measured by the Griess reaction (Green etal., 1982), whereas nitrates were first reduced by nitrate reduc-tase (EC 1.6.6.2.) from Aspergillus (Boehringer Mannheim) inthe presence of NADPH and FAD (Bories and Bories, 1995).Total nitrites (reduced NO3

– and preexisting NO2–) were eval-

uated spectrophotometrically at 546 nm and considered as totalstable metabolites of NO. Sodium nitrite and nitrate solutions(Merck) were used for preparing the standard solutions. Theresults are expressed in units of �mol/g wet weight of tissue.

Evaluation

Histochemistry and immunohistochemistry. Theboundaries of the different regions were determined, and ND-stained cells were counted by the same author (B.B.). Theseplanimetric analyses were performed in three consecutive sec-tions from the same series (150 �m apart), corresponding toBregma levels ranging from 1.20 to 1.70 mm (Paxinos andWatson, 1998). Rectangular frames (200 � 215 �m) werecaptured with a 20� objective and a color video camera (JVCTK-1280E), and positive neurons were counted semiautomat-ically in each frame. ND-positive neurons were counted onlywhen second-order dendrites were distinguished. In total 81frames from cortex (in three well-defined axes), 48 from corpusstriatum (caudate putamen nucleus, avoiding edges with externalcapsule), and 37 from accumbens nucleus (rostral, central, andcaudal parts) were analyzed for each animal. This procedureallowed us to compare the total numbers of ND/NOS-positivecells 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, asdescribed above, the percentage of cells for each saline- ornicotine-treated animal was calculated relative to that of naıveanimals of the same sex, and the statistical evaluation was carriedout accordingly: ANOVAs were performed to determine thevariance, with sex (male, female) and treatment (saline, nicotine)as factors.

Nitrite and nitrate determinations. In the statisticalanalysis of the biochemical determinations, ANOVAs were em-ployed, with sex (male, female), treatment (naıve, saline, nico-tine), and brain regions (cortex, striatum, accumbens nucleus) asthe factors and NO3

–/NO2– levels as the dependent variable.

Duncan’s post hoc analyses were performed as required. Toallow for comparisons between the results obtained in the bio-chemical and histological analyses, the results from both types ofexperiment are represented as percentages of the naıve animals’values for each saline- or nicotine-treated group.

RESULTS

HistologyNeuronal NOS immunoreactivity and ND activity

colocalized in the same population of neurons in the threeregions were studied after double-labelling analyses (notshown) and comparing both types of staining (Fig. 1).However, the stain obtained with the histochemical tech-nique provided more details about the morphology of thepositive elements than the immunohistochemical proce-dure, i.e., a higher degree of branching. Therefore, weemployed ND staining for quantitative analyses. Positiveneurons in the three regions studied were seen to corre-spond to type I ND elements and mainly had multipolarbranching dendrites (Fig. 1).

Because the experiments were carried out in groupsof six animals, one animal from each group, data evalua-tion was carried out accordingly. The ND/NOS-positiveneuron counts obtained from each brain region ofnicotine- or saline-treated rats were represented as thepercentages of the counts for naıve animals for each sexseparately. Figure 2 depicts the percentage difference from

Nicotine and Nitric Oxide Synthase 691

Fig. 1. Adjacent coronal sections from a male naıve rat immunolabelledfor neuronal NOS (nNOS; A,C–E) or stained with the histochemicalreaction for NADPH-diaphorase (ND; B,F–H). A,B: Panoramic viewsof the central rostrocaudal level where quantitative biochemical andhistochemical analyses were performed. Details from the three regionsstudied, 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 bloodvessels, which was more patent in the cerebral cortex (see B,F). How-ever, at higher magnification, ND-stained neural elements (F–H) aremore easily distinguishable compared with immunolabelling (C–E).Both types of techniques employed yield the staining of similar neu-ronal populations, with similar cell densities in each region. Scalebars � 1 mm for A,B, 50 �m for C–H.

naıve male and female rats in groups that received saline ornicotine injections. Multifactorial ANOVA revealed a sig-nificant main effect of treatment [F(2,89) � 3.29; P �0.043], implying a difference among naıve, saline-, andnicotine-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 inthe corpus striatum of female rats, where saline injectionslowered the number of ND/NOS-positive neurons (85%of naıve). Figure 3 shows the corpus striatum of femalesfrom the three experimental groups. Table I gives bothnumber and cell densities in the three brain regions stud-ied.

BiochemistryA multifactorial ANOVA with sex (male, female),

treatment (naıve, saline, or nicotine), and brain region(cortex, striatum, accumbens) as the factors and NO2

–/NO3

– levels as the dependent variable revealed significant

main effects of brain regions [F(2,102) � 7.36; P 0.001]and treatment [F(2,102) � 43.18; P 0.0001] and aninteraction between brain regions and treatment[F(4,102) � 6.21; P 0.0001]. As depicted in Figure 4nicotine caused a significant increase in NO2

–/NO3– lev-

els in all brain regions in both sexes compared with thenaıve or saline-treated animals. Post hoc analyses, inaddition to showing that nicotine caused a significantincrease in all regions tested, also revealed that salineinjections were not inert and lowered cortical NO2

–/NO3

– levels in female rats: The saline-treated females hadsignificantly lower cortical NO2

–/NO3– levels than naıve

females.Figure 5 shows the percentage difference in naıve

male and female rats in the groups that received saline ornicotine injections. Comparison of Figure 5 (biochemis-try) with Figure 2 (histochemistry) reveals that the re-sponse to nicotine as measured by changes in brain NO2

–/NO3

– levels is substantially higher than that measured bynNOS histochemical expression.

DISCUSSIONIn the present work an increase in the production of

NO in three different brain regions (frontal cortex, corpusstriatum, and accumbens nucleus) was seen after repetitiveexperimental administration of nicotine (chronic treat-ment), as reflected by an increase in the levels of nitratesand nitrites. Moreover, this is the first time a comparisonis offered concerning the effect of nicotine on NO pro-duction using both biochemical and histological ap-proaches, in that the ND histochemical expression wasanalyzed in neurons of the same brain regions in parallel.The histochemical and immunohistochemical studies ofND/NOS-positive neurons also revealed a similar trend;however, the magnitude of the response was not nearly ashigh as that reflected in NO2

–/NO3– levels. The results of

the present study confirm the observations of a previousstudy by Pogun et al. (2000) and show that chronicnicotine treatment in rats increases the levels of stablemetabolites of NO in both male and female brains.

One aspect of this study was the demonstration ofdiminished NOS expression because of the stress inducedby chronic injections. There was a parallel variation in NOmetabolites. In both measurements, this effect reachedsignificant levels in female rats. This finding supports theview that NO may be involved in the stress response (forreview see Lopez-Figueroa et al., 1998). In studies carriedout under diverse stressors employing biochemical meth-ods or the ND histochemical technique (Kishimoto et al.,1996; Vaid et al., 1997; Sanchez et al., 1999; Veenman etal., 1999), increases in the NOS activity and/or expressionhave been reported in the limbic-hypothalamic-pituitary-adrenal axis. Thus, we describe for the first time a sexuallydimorphic change in NOS expression and activity inresponse to chronic injections out of the stress axis (i.e.,the striatum and cerebral cortex).

The effects of nicotine on the nitrergic system werenot the same in both sexes. The results obtained are inagreement with other animal studies in which male and

Fig. 2. Percentage difference of the number of ND/NOS-positiveneurons in nicotine- or saline-treated rats compared with naıve animalsin the three regions studied in male and female rats. Bars representmeans SEM. The asterisk indicates a statistically significant differencefrom naıve rats, same sex, same brain region (Duncan’s post hoc t-test,P � 0.017).

Nicotine and Nitric Oxide Synthase 693

female rodents were found to show different sensitivitiesto the effects of nicotine. For example, food intake andphysical activity (Bowen et al., 1986; Grunberg 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 etal., 1999) display sexual dimorphism when progressingunder the influence of nicotine. Despite the huge differ-ences in the experimental animal used; the dose, route,and duration of the drug administration; and the biobe-havioral task employed, the evidence is that nicotine exertsmost of its influence on brain physiology following asexually dimorphic pattern. The mechanisms underlyingthis sex difference in nicotine effects, including thoserelated to brain NO production, remain uncertain, butrecent in vivo and in vitro evidence strongly suggests thatsex hormones can modulate the activation of the �4�2neuronal acetylcholine receptors and the blockage of an-tinociception by nicotine (Damaj, 2001).

Our results indicate significant differences in theresponse to chronic treatment with nicotine on measuringNO metabolites levels and the number of neurons positivefor NOS/ND. There are at least three possible explana-tions for this divergence. First, the immunohistochemicaland histochemical localization of NOS/ND within eachpositive cell is not a quantitative indicator of NO produc-tion: With these methods the presence of the enzyme orits activity is detected, but they fail to demonstrate howmuch NO is actually produced. It is therefore possible thatthere was indeed an increase in the NO stable metabolitesafter nicotine injection (produced by the same NOS/ND-positive neurons) but no de novo expression of the en-zyme, the number of counted positive neurons thus in-creasing. The degree of staining of ND-positive neuronsmay be related to the level of NOS enzyme activation(DellaCorte et al., 1995; Morris et al., 1997; Weruaga etal., 2000), but the former histological quantification is notreliable enough to ensure that the positive neuronscounted produce more neurotransmitter. Second, the mis-match between the biochemical and the histochemicalresults may be due to production of NO from sourcesother than neurons. Nonactivated astrocytes are an impor-tant site of nNOS, but its histological demonstration mustbe made either with special tissue fixation and freezing orwith weak formaldehyde fixation (Gabbott and Bacon,1996; Kugler and Drenckhahn, 1996), which is, on theother hand, incompatible with both the discrimination ofneuronal elements and the quantification of their densityin large brain regions. ND staining related to inducibleNOS (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 naıve (A), saline-injected (B), and nicotine-injected (C) female ratson sections stained histochemically for NADPH-diaphorase. Quanti-tative analyses of stained cells densities demonstrated a statisticallysignificant decrease only in the saline group compared with the naıve(untreated) or the nicotine-injected animals. Scale bar � 100 �m.

694 Weruaga et al.

astrocytes or microglial cells in diverse brain insults (Lei etal., 1996; Wallace, 1996; De Groot et al., 1997; Ding etal., 1997; Wallace et al., 1997; Stojkovic et al., 1998).However, to our knowledge, there is no documentationof enhanced NO production from glial cells resulting fromnicotine treatment. Because nicotinic receptors are func-tional in astrocytes (Hosli et al., 1997, 2000), studies arecurrently being undertaken to clarify this issue.

Finally, another source of NO is blood vessels, whichcontain endothelial NOS (eNOS; NOS III; for review seeMoncada, 1997). As shown in Figure 1, ND histochemicalstaining labels blood vessels. This staining corresponds toeNOS rather than nNOS. However, the histologicalquantification of these is probably not as feasible asneuronal staining. Recent evidence has been foundsupporting this possibility: Using the L-[3H]arginine andL-[3H]citrulline assay and different enzyme preparations,Tonnessen et al. (2000) have demonstrated that nicotineincreases the activity of eNOS more than that of nNOSand has no effect on inducible NOS. Thus, it is morelikely that nicotine would enhance NO2

–/NO3– levels

mainly through the eNOS located in blood vessels ratherthan through the neuronal isoform detected by histolog-

ical techniques. Pharmacological assays using selectiveNOS isoform inhibitors could be used to test this hypoth-esis.

It may be concluded that, although nicotine causesan overall increase in NOS positive neurons, the stressarising from chronic injections reduces the number ofNOS-positive neurons and that this latter effect is mostprominent in the female corpus striatum. Biochemical

TABLE I. Number and Density of NOS/ND-Positive Cells*

Cortex Striatum Accumbens

Cell count Density Cell count Density Cell count Density

MaleNaıve 27.4 3.2 17.2 2.0 103.4 9.9 65.0 6.2 71.0 3.5 44.6 2.2Control 28.6 5.1 18.0 3.2 91.0 6.9 57.2 4.4 69.4 7.9 43.6 4.9Nicotine 33.4 3.7 21.0 2.4 96.6 4.7 60.7 3.0 78.8 2.3 50.0 1.4

FemaleNaıve 29.5 2.9 18.5 1.8 102.8 6.5 64.6 4.1 70.2 7.3 44.1 4.6Control 25.6 2.6 16.1 1.6 87.2 5.0 54.8 3.2 59.8 4.9 37.6 3.2Nicotine 26.4 3.6 16.6 2.3 98.6 6.9 62.0 4.3 70.2 5.7 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- orsaline-treated rats compared with naıve animals. Bars are grouped bybrain regions and represent means SEM. Symbols on the barsrepresent different degrees of statistical significance for differences be-tween naıve 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 andin the same brain region, as post hoc Duncan’s analyses.

Fig. 5. Percentage difference of NO2–/NO3

– levels in nicotine- orsaline-treated rats compared with naıve animals in the three regionsstudied in male and female rats. Bars represent means SEM. For posthoc statistical evaluation, see Figure 4.

Nicotine and Nitric Oxide Synthase 695

assays of NO2–/NO3

– in rat brain following nicotinetreatment reveal a greater level of NO production com-pared with the number of NOS/ND-positive neurons,suggesting the involvement of eNOS and/or other non-neuronal sources of nNOS.

ACKNOWLEDGMENTSThe authors thank Dr. Dilek Taskiran for her guid-

ance in the biochemical assays. The help of N. Skinner inrevising the English version of the manuscript is acknowl-edged.

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