Sex differences in catechol contents in the olfactory bulb of ...Sex differences in catechol contents in the olfactory bulb of control and unilaterally deprived rats C. Go´mez,1 J

Sex differences in catechol contents in the olfactorybulb of control and unilaterally deprived rats

C. Gomez,1 J. G. Brinon,1 J. Valero,1 J. S. Recio,1 A. R. Murias,1 G. G. Curto,1 L. Orio,2 M. I. Colado2 and J. R. Alonso1

1Laboratory Plasticidad Neuronal y Neurorreparacion, Instituto de Neurociencias de Castilla y Leon, Universidad de Salamanca,E-37007 Salamanca, Spain2Departamento de Farmacologıa, Facultad de Medicina, Universidad Complutense, E-28040 Madrid, Spain

Keywords: 3, 4-dihydroxyphenylacetic acid, dopamine, homovanillic acid, olfactory deprivation, sexual dimorphism, tyrosinehydroxylase

Abstract

The dopaminergic system plays important roles in the modulation of olfactory transmission. The present study examines thedistribution of dopaminergic cells and the content of dopamine (DA) and its metabolites in control and deprived olfactory bulbs (OB),focusing on the differences between sexes. The content of DA and of its metabolites, dihydroxyphenylacetic acid (DOPAC) andhomovanillic acid (HVA), were measured by HPLC. The morphology and distribution of dopaminergic neurons were studied usingtyrosine hydroxylase (TH) immunohistochemistry. Cells were typified with TH–parvalbumin, TH–cholecystokinin or TH–neurocalcindouble-immunofluorescence assays. Biochemical analyses revealed sex differences in the content of DA and of its metabolites. Innormal conditions, the OBs of male rats had higher concentrations of DA, DOPAC and HVA than the OBs of females. Theimmunohistochemical data pointed to sex differences in the number of TH-immunopositive cells (higher in male than in female rats).Colocalization analyses revealed that dopaminergic cells constitute a different cell subpopulation from those labelled afterparvalbumin, cholecystokinin or neurocalcin immunostaining. Unilateral olfactory deprivation caused dramatic alterations in thedopaminergic system. The DA content and the density of dopaminergic cells decreased, the contents of DA and DOPAC as well asTH immunoreactivity were similar in deprived males and females and, finally, the metabolite ⁄ neurotransmitter ratio increased. Ourresults show that the dopaminergic modulation of olfactory transmission seems to differ between males and females and that it isregulated by peripheral olfactory activity. A possible role of the dopaminergic system in the sexually different olfactory sensitivity,discrimination and memory is discussed.

Introduction

Dopamine (DA) is one of the most abundant neurotransmitters in themain olfactory bulb (MOB). Subpopulations of juxtaglomerularinterneurons and small populations of neurons in the remaining layersexpress tyrosine hydroxylase (TH; the rate-limiting enzyme in the DAsynthesis pathway; Halasz et al., 1981). In the glomerular layer (GL),TH colocalizes with GABA but not with other neurochemical markers(Kosaka et al., 1998). Physiological studies suggest that DA caninhibit activation of the main projection neurons (Berkowicz &Trombley, 2000; Davila et al., 2003). The DA inhibits synaptictransmission between olfactory sensory neurons and projectionneurons (Ennis et al., 2001), presumably controlling the GABAergicinhibitory processing of odour signals (Brunig et al., 1999). Wilson &Sullivan (1995) have proposed that TH-positive neurons maymodulate lateral and feedback inhibition among glomeruli in theMOB. The magnitude and varied locations of dopamine modulation inthe MOB indicate an important role for DA in odourant signalprocessing.

Important degrees of sexual dimorphism have been found incatecholaminergic neurotransmission in the mammalian central

nervous system (Vaccari et al., 1977, 1980). As examples, thedistribution of TH-immunoreactive (-IR) elements in the anteroventralperiventricular nucleus of rats, the regulation of TH gene in the micelocus coeruleus, and DA transporter activity in the striatum of mice aresexually differentiated (Simerly et al., 1985; Thanky et al., 2002;Bhatt & Dluzen, 2005).Olfaction is sexually dimorphic and, for example, women outper-

form men in odour memory (Oberg et al., 2002). Sex differences in theolfactory pathway have mainly been described in the accessoryolfactory system, which responds to sex-differentiated pheromones(Segovia & Guillamon, 1993; Luo et al., 2003) and is involved in thecontrol of reproductive behaviour (Guillamon & Segovia, 1997). Littleevidence exists to suggest sexual dimorphism in the structure of themain olfactory system (Gomez et al., 2005), although some specifictypes of olfactory behaviour mediated by this system, such as therecognition of volatile odours, are sexually dimorphic in mice (Baum& Keverne, 2002; Keverne, 2004), suggesting sex differences in thestructure and function of the main olfactory system.TH expression and the DA content in the MOB depend on

peripheral stimulation (Baker, 1990; Brunjes, 1994). In fact, of all thealterations occurring in the MOB of rodents after the loss of functionalinput, the most dramatic changes are found in the catecholaminergicsystem. Olfactory deprivation causes a decrease in the number of TH-IR juxtaglomerular cells (Brunjes, 1994), a profound reduction in

Correspondence: Dr Jose R. Alonso, as above.E-mail: [email protected]

Received 15 September 2006, revised 8 January 2007, accepted 9 January 2007

European Journal of Neuroscience, Vol. 25, pp. 1517–1528, 2007 doi:10.1111/j.1460-9568.2007.05407.x

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

juxtaglomerular TH mRNA (Stone et al., 1991; Baker et al., 1993), adecrease in DA and 3,4-dihydroxyphenylacetic acid (DOPAC) levels(Philpot et al., 1998), and an increase in dopamine D2 receptor density(Guthrie et al., 1991).Based on the following four postulates, (i) that the neurochemistry

of dopaminergic cells in the infraglomerular layers is not known;(ii) that the catecholaminergic system exhibits sexual dimorphism;(iii) that olfactory function is sexually dimorphic; and (iv) that thecatecholaminergic elements in the olfactory system respond toalterations in afferent activity, we investigated whether the contentof DA and of its metabolites, DOPAC and homovanillic acid (HVA),as well as TH expression, differed in the olfactory bulb (OB) of malesand females. Moreover, we performed a characterization of dopam-inergic cells in the infraglomerular layers. These analyses were carriedout both under control conditions and 60 days after unilateralolfactory deprivation.

Materials and methods

Animals and neonatal naris occlusion

Pregnant female Wistar rats were housed singly with food and wateravailable ad libitum. On the day of birth (P0), rat pups wereanaesthetized by hypothermia and underwent either unilateral narisclosure through a brief cautery to one external naris (deprivedcondition), or a sham operation with identical treatment, except thatthe cautery was placed on the top of the nose. Normal body temperaturewas then restored by placing the pups under a heating lamp. The lesionswere examined daily, and only those animals showing complete closureof the naris in all examinations were used for the analyses. In all cases,the experimental procedures conformed to NIH guidelines and were inaccordance with the European Community Council Directive(86 ⁄ 609 ⁄ EEC) and current Spanish legislation for the use and careof laboratory animals (BOE 252 ⁄ 34367–91, 2005)

Immunohistochemistry

Eight unilaterally deprived Wistar rats (four males and four females)and 16 naıve animals, which did not undergo surgery (eight males andeight females), were perfused when they reached P60. They weredeeply anaesthetized with 1 mL ⁄ kg body weight of a mixture of 3 ⁄ 4ketamine hydrochloride (Ketolar; Parke-Davis, Barcelona, Spain) and1 ⁄ 4 tiacine hydrochloride (Rompun, Bayer, Leverkussen, Germany).The animals were then perfused intra-aortically with 100 mL of 0.9%saline followed by 300 mL of a fixative composed of 4% (w ⁄ v)paraformaldehyde and 0.2% (w ⁄ v) picric acid in 0.1 m phosphatebuffer, pH 7.4 (PB). After perfusion, the brains were removed and theOBs dissected out and postfixed for 2 h in the same fixative. Then,tissue blocks were washed in PB, cryoprotected with 15% (w ⁄ v)sucrose for 12 h at 4 �C, and immersed in 30% (w ⁄ v) sucrose untilthey sank. After cryoprotection, 30-lm coronal sections were cut on afreezing sliding microtome (Leica Jung SM2000; Nussloch,Germany). The sections were collected in six series in 0.05% sodiumazide (w ⁄ v) in PB at 4 �C.The immunohistochemical technique was carried out on coronal

serial sections following the avidin–biotin–peroxidase method (Hsuet al., 1981). Sections were rinsed in PB (3 · 10 min) and sequen-tially incubated in: (i) 1 : 10 000 rabbit anti-TH (Jacques Boy, no.208020234; Reims, France) in PB with 0.2% Triton X-100 for 48 h at4 �C; (ii) 1 : 200 biotinylated goat antirabbit IgG (Vector, Burlingame,CA, USA) in PB for 1 h at room temperature; and (iii) 1 : 200Vectastain Elite ABC reagent (Vector) in PB for 1 h at room

temperature. Between successive steps the sections were carefullyrinsed in PB (3 · 10 min). The reaction product was visualized byincubating the sections in 0.025% 3,3¢-diaminobenzidine and 0.003%hydrogen peroxide in PB until the desired staining intensity wasreached.

Double-immunofluorescence histochemistry

Coronal sections were rinsed in 0.1 m phosphate-buffered saline(PBS), pH 7.4, for 3 · 10 min and then incubated sequentially in:(i) 1% sodium borohydride in PBS for 20 min at room temperature;(ii) 0.2% Triton X-100 and 5% normal goat serum in PBS for 1 h atroom temperature; (iii) 1 : 10 000 rabbit anti-TH (Jacques Boy, no.208020234) and either 1 : 3000 mouse antiparvalbumin (PV; Clone235, Celio et al., 1988), or 1 : 600 mouse anticholecystokinin (CCK;provided by CURE ⁄ Digestive Diseases Research Center, Anti-body ⁄ RIA Core, University of California, Los Angeles, USA) or1 : 7000 mouse anti-TH (Incstar, no. 22941; Stillwater, MN, USA)and 1 : 500 rabbit antineurocalcin (NC; Biomol International LP, no.NL 3770; Plymouth Meeting, PA, USA), in PBS with 0.2% TritonX-100 and 2.5% normal goat serum for 48 h at 4 �C; (iv) 1 : 200Cy2-conjugated goat antirabbit IgG (Jackson ImmunoResearch, no.111-225-003; West Grove, PA, USA) and 1 : 200 Cy3-conjugatedgoat antimouse IgG (Jackson ImmunoResearch, no. 115-165-166) inPBS for 2 h at room temperature; and (v) 1 : 2000 propidium iodide(PI; Sigma) in PBS for 30 min. The PI counterstaining allowed us todistinguish immunonegative cells in the MOB. Between successivesteps the sections were carefully rinsed in PBS (3 · 10 min). Finally,sections were examined with both a microscope (Olympus ProvisAX70, Tokyo, Japan) equipped with epifluorescence and appropriatefilter sets and a confocal microscope (Leica TCS SP2, Mannheim,Germany).

High-performance liquid chromatography (HPLC)

Ten unilaterally deprived Wistar rats (five males and five females),four sham-operated animals (two males and two females) and sixnaıve animals (three males and three females) with identical ages(P60) were killed by cervical dislocation and decapitation. Their brainswere rapidly removed and the OBs dissected out on ice. The tissuewas homogenized and DA, DOPAC and HVA were measured byHPLC with electrochemical detection. The mobile phase consisted ofKH2PO4 (0.05 m), octanesulphonic acid (1 mm), EDTA (0.1 mm) andmethanol (16%) and was adjusted to pH 3.0 with phosphoric acid. Theflow rate was 1 mL ⁄ min. The HPLC system consisted of a pump(Waters 510) linked to an automatic sample injector (Loop 200 lL,Waters 717 plus Autosampler), a stainless steel reverse-phase column(Spherisorb ODS2; 5 lm, 150 · 4.6 mm) with a precolumn and acoulometric detector (Coulochem II; Esa Inc, Chelmsford, MA, USA).The working electrode potential was set at 400 mV. The currentproduced was monitored by means of integration software (Unipoint,Gilson, Middleton, WI, USA).

Image analysis

Quantification of the number of TH-IR somata was carried out usingNeurolucida and Neuroexplorer software (MicroBrightField, Colches-ter, VT, USA) at the same rostrocaudal levels of the six groups ofMOBs (deprived, contralateral and control OBs from males andfemales) (Fig. 1). All analyses were carried out choosing sectionswith specific neuroanatomical landmarks that made them easily

1518 C. Gomez et al.

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 25, 1517–1528

distinguishable among animals (Fig. 1). The GL was delimited, themiddle point of the section was marked and from it a vertical lineextending 0.75 mm above and below it was drawn (see Fig. 1b fordetails). The ends were projected to the medial region of the GL,obtaining an objectively measurable surface in the GL. Eachjuxtaglomerular cell immunopositive for TH located inside the drawnarea was included in the analysis. Data are given as cells ⁄ mm2.

The number of positive cells in the remaining layers was muchlower, and hence all the cells in the whole of each layer were analysed.The analysis in the infraglomerular layers was carried out on foursections per animal, separated from each other by 360 lm (Fig. 1a).One line throughout the mitral cell layer and another throughout theinterface between the external plexiform layer (EPL) and the GL weredrawn in each section. Each TH-IR cell in the infraglomerular layersof each section was included in the analysis, differentiating thoselocated in EPL from those located in the remaining layers (mitral celllayer and inframitral layers). The areas of the EPL and inframitrallayers were measured and the density of TH-IR cells was obtained foreach region as the number of cells ⁄ mm2. Finally, the mean of thedensity in the four sections was obtained for each animal.

The study of the accessory olfactory bulb (AOB) was performed in48 sections (a rostral and a caudal level for each animal; Fig. 1c), inwhich the surface of the AOB was delimited and intensely stainedTH-IR cells were counted (Fig. 1c). The results are expressed asnumber of cells ⁄ mm2.

Statistical analysis

The SPSS statistical software program (SPSS, Chicago, IL, USA) wasused. One-way anova followed by Dunnett’s test were performed forthe comparison of DA, DOPAC and HVA contents as well as thenumber of TH-IR cells between deprived, contralateral and controlOBs while Student’s t-test was implemented for sex comparisons.Because unilateral olfactory deprivation causes a decrease in thevolume of the ipsilateral MOB, the effect of size reduction wasconsidered and a second group of densities corrected for this shrinkageeffect was also analysed in all comparisons including ipsilateral MOB.The data are expressed with respect to both uncorrected and correctedconditions, and the consequences of size reduction were evaluated foreach statistical analysis. Mean values are quoted ± SEM.

Results

TH-IR cell distribution and morphology

Immunohistochemical analyses in the MOB showed that TH-IR cellswere mainly located in the GL. Most of the immunolabelledjuxtaglomerular neurons were small, with spherical somata and oneintraglomerular dendrite, and were identified as periglomerular cells(Fig. 2a), although a number of larger cells was also found in this layer(Fig. 2a). In addition, TH was also localized within small and largesomata located in the EPL and in the mitral and inframitral layers

Fig. 1. Neurolucida drawings of representative coronal sections of the OB where the number of dopaminergic cells was analysed. (a) TH-IR cells in the EPL (n)and in the mitral and inframitral layers (s). (b) Dopaminergic cells in the medial region of the GL (n). (c) Catecholaminergic cells in the AOB (m). AOB,accessory olfactory bulb; AON, anterior olfactory nucleus; EPL, external plexiform layer; GL, glomerular layer. Scale bars, 1 mm (a–c), 100 lm (inset in b).

Dopaminergic system in the olfactory bulb 1519


Fig. 2. Morphological diversity of dopaminergic neurons in the MOB and AOB. (a) TH-IR cells in the GL of the MOB. White arrowheads point to small andspherical juxtaglomerular neurons and black arrows show larger cells in the same layer. (b–e) Different morphological types of dopaminergic cells found in the EPL.(f) Dopaminergic cells in the granule cell layer had small somata with a thick apical prolongation. (g and h) TH-IR cells in the AOB. Some (g) had large somatawith several prolongations and complicated ramifications whereas others (h) possessed small somata with few prolongations. Scale bar, 50 lm.



(Fig. 2). Although the identification of these cells is more uncertain,larger TH-IR cells in the GL and EPL exhibited the morphologicalfeatures of interneurons, presumably short-axon cells (see Discussion).In addition to somata, a high density of TH-IR neuropil was alsoobserved. These prolongations belonged not only to bulbar dopam-inergic cells but also to cells located in the locus coeruleus that sendtheir axons to the MOB, constituting the noradrenergic centrifugalmodulatory system.

While the number of TH-IR neurons in the MOB was high, thenumber found in the AOB was much lower. Some of the TH-IR cellsin the AOB were only slightly immunostained; they were small andhad spherical somata. Other cells, intensely stained, were largeand were mainly located surrounding the olfactory glomeruli (Fig. 2gand h). A 30-lm section of a male AOB contained a mean ± SEM of3.43 ± 0.87 intensely stained TH-IR cells, i.e. a density of6.56 ± 0.93 cells ⁄ mm2, whereas the TH-IR juxtaglomerular celldensity in the GL of the male MOB was 1320.28 ± 45.27 cells ⁄ mm2.In females, the density of TH-IR cells in the AOB was9.01 ± 1.12 cells ⁄ mm2 while in the GL of the MOB it was1226.98 ± 39.76 cells ⁄ mm2. These data are a good indication of thedifferences in the intrinsic dopaminergic modulation occurring in theMOB and in the AOB.

Sexual dimorphism

In control animals, HPLC analysis revealed sex differences in thecatechol content of the OB, males showing higher concentrations ofDA, DOPAC and HVA than females (Fig. 3a). The statistical analysesrevealed that the DA content was � 30% higher in males than infemales (Student’s t-test, P < 0.01; Fig. 3a), the DOPAC content was50% higher in males (Student’s t-test, P < 0.01; Fig. 3a), while theHVA content showed the highest difference, being 65% higher inmales than in females (Student’s t-test, P < 0.01; Fig. 3a). Thus, theamounts of DA as well as its degradation metabolites differ betweenmales and females.

In parallel to the HPLC data, the immunohistochemical analysesrevealed sex differences in the density of cells immunopositive for TH,this sexual dimorphism being dependent on cell distribution. Although

the male MOBs had more TH-IR cells ⁄ mm2 than female MOBs in allregions analysed (Fig. 3b), this difference was only significant in theEPL (29.51 ± 1.43 in males vs. 26.12 ± 0.98 cells ⁄ mm2 in females;Student’s t-test, P < 0.05; Fig. 3b); the layer with the lowest differencewas the GL (1320 ± 45 in males vs. 1226 ± 39 cells ⁄ mm2 in females;Student’s t-test, P ¼ 0.229). Contrary to the EPL of the MOB, in theAOB the density of TH-IR cells was similar in the two sexes in thatthe differences were not statistically significant (5.71 ± 1.39 in malesvs. 6.64 ± 1.63 cells ⁄ mm2 in females; Student’s t-test, P ¼ 0.677).Dopaminergic cells were typified by comparison with other cell

subpopulations with a morphology and distribution similar to TH-IRcells. Sections processed for double-immunofluorescence stainingwith TH and antisera against PV (Fig. 4a–d), NC (Fig. 4e–h) and CCK(Fig. 4i–l) were analysed by confocal microscopy to assess thecolocalization of TH with a marker of interneurons of the EPL (PV), asexually dimorphic marker in the MOB of mice (NC) or with a peptideexpressed by many tufted cells (CCK). TH-IR cells in the EPL werenot immunoreactive for any of the other markers (PV, NC and CCK).Although all these markers were highly expressed by EPL neurons andalthough some had similar morphological features to TH-IR cells,bulbar dopaminergic neurons constitute subclasses of interneuronsdifferent from those containing PV, NC or CCK.

Effects of unilateral olfactory deprivation in the numberof TH-IR cells

60 days of unilateral deprivation caused a decrease in the number ofcells immunostained for TH in the ipsilateral OB (Figs 5 and 6). Nodifferences were detected between contralateral and the correspondingcontrol OBs. The statistical analyses showed that the decrease in thenumber of TH-IR cells was not only observed in the GL, where it wasclearly evident and has been previously reported, but also in the EPL(Fig. 6). Although the TH-IR cell density increased after deprivation inboth the EPL and the inframitral layers, when the correction factor forthe area reduction was applied in the ipsilateral MOB, the differenceswere no longer statistically significant (in the case of inframitral layersof females; Fig. 6b) or showed a decrease in the number of TH-IRcells (in the case of the EPL in males). After the correction of the data

Fig. 3. Sex comparison of (a) DA, DOPAC and HVA concentrations (n ¼ 10, five males and five females) and of (b) TH-IR density (n ¼ 16, eight males andeight females) of control OBs. Results shown as mean ± SEM. Asterisks indicate significant differences in the catechol content or in the number ofTH-IR cells ⁄ mm2 between males and females. *P < 0.05, **P < 0.01.



Fig. 4. Confocal images [17.1 lm stack for (a)–(d), 14.82 lm stack for (e)–(h) and 10.83 lm stack for (i)–(l)] of double immunofluorescences contrasted withPI coronal sections from control MOBs to analyse the colocalization of TH with (a–d) PV, (e–h) NC or (i–l) CCK. (a, e and i) PI staining in three different sections.(b, f and g) Cells containing TH (arrows). (c, g and k) PV- (c), NC- (g) and CCK (k)-immunoreactive cells in the EPL (solid arrows). (d, h and i) Overlays of thethree pictures show that no TH-IR cells were immunopositive for (d) PV, (h NC) or (l) CCK. Scale bar, 75 lm.

Fig. 5. TH immunostaining of equivalent regions from (a) a control MOB and (b) a MOB ipsilateral to naris occlusion. Note the reduction in the number ofdopaminergic cells in the GL and in the EPL of the deprived MOB and the increase in the TH-IR fibre density in the infraglomerular layers. Scale bar, 100 lm.



for the volume shrinkage, the density of TH-IR cells in the EPL ofmales fell by � 50% (29.51 ± 1.43 cells ⁄ mm2 in control vs.16.41 ± 1.70 cells ⁄ mm2 in the ipsilateral ‘corrected’ MOB; mean ± -SEM, Dunnett’s test, P < 0.05; Fig. 6a). The decrease in females wasless marked and was not statistically significant (Fig. 6c). Moreover,the number of dopaminergic cells ⁄ mm2 in the different regions of thedeprived MOBs was similar in the two sexes, unlike what wasobserved in control animals. While control MOBs from male animalsshowed higher dopaminergic cell density (Fig. 3b), after deprivationthe number of TH-IR cells ⁄ area in the EPL was not significantlydifferent between males and females (34.24 ± 4.23 cells ⁄ mm2 indeprived males vs. 35.91 ± 2.48 cells ⁄ mm2 in deprived females;mean ± SEM, Student’s t-test, P ¼ 0.487; Fig. 7b).

In addition to the reduction in the number of TH-IR somata, anincrease in the TH-IR fibre density was quite patent (Fig. 5). Fibrespositive for TH are also dopamine-b-hydroxylase-immunoreactive and

therefore belong to the noradrenergic centrifugal system (Gomezet al., 2006). Previous studies have shown that noradrenergic fibredensity increases after unilateral olfactory deprivation (not only due tothe reduction of the bulbar area but also because of the lack ofstimulation) and this was also observed in this study after THimmunohistochemistry (Fig. 5).

Effects of the unilateral olfactory deprivation on catecholconcentration

The reduction in the number of TH-IR neurons was correlated with adecrease in DA content (Fig. 6d). Ipsilateral OBs from both males andfemales showed similar reductions in their DA content. The DAconcentration in control OBs from males was 77.61 ± 4.41 ng ⁄ g andthis was reduced to 33.55 ± 4.22 ng ⁄ g in ipsilateral male OBs(Dunnett’s test, P < 0.01). The females showed reductions of 56%

Fig. 6. (a–c) TH-IR cell density and (d) DA content 60 days after unilateral olfactory deprivation. (a and b) TH-IR cell density in different layers of the MOB of(a) males and (b) females. White barrels show control MOB, grey barrels contralateral, black barrels ipsilateral and banded barrels corrected ipsilateral to olfactorydeprivation. Because odour deprivation decreases the overall size of the MOB, the TH-IR density might simply arise from general shrinkage of this bulbar region. Toexplore this possibility, we normalized TH density with respect to the reduction in size of the MOB. ‘Corrected ipsilateral’ refers to data from deprived MOB,adjusted for area shrinkage in the layers studied. (c) Number of TH-IR cells ⁄ mm2 in control, contralateral, ipsilateral and corrected ipsilateral EPLs of males andfemales. (d) DA concentration in the different groups of OBs in males and females. Results are shown as mean + SEM. *P < 0.05, **P < 0.01 for contralateral,ipsilateral or corrected ipsilateral OBs vs. control OBs.



(59.17 ± 3.51 ng ⁄ g in controls vs. 25.82 ± 2.93 ng ⁄ g in the ipsilat-eral OB; mean ± SEM, Dunnett’s test, P < 0.01).While control OBs from male animals showed significantly higher

levels of the three compounds analysed by HPLC (DA, DOPAC andHVA) than females, after deprivation DA and DOPAC levels did notsignificantly differ between males and females (Fig. 7a). AlthoughHVA continued to be higher in males than in females, the sexdifferences decreased (74.06 ± 11.35 ng ⁄ g vs. 39.32 ± 3.03 ng ⁄ g;mean ± SEM, Student’s t-test, P < 0.05). Thus, the sex differences inDA and DOPAC found under control conditions were no longerobserved after olfactory deprivation.After deprivation, DA metabolism differed in the ipsilateral OBs of

males and females (Fig. 8). The response of DA metabolites underolfactory deprivation, namely under the reduction of the DA content,differed between males and females. While HVA levels seemed to actsimilarly in males and females, DOPAC did not (Fig. 8a and b). Inmales, the DOPAC concentration decreased with the decrease in DAcontent (50.36 ± 2.08 ng ⁄ g in control males vs. 25.80 ± 4.05 ng ⁄ g inthe ipsilateral OB of deprived male rats; mean ± SEM, Dunnett’s test,P < 0.01; Fig. 8a). However, no differences were detected in theDOPAC content between the three groups of OBs in females (control,contralateral and ipsilateral; Fig. 8b). Female control OBs showedconcentrations of 33.11 ± 2.57 ng ⁄ g, contralateral OBs 33.9 ± 4.09and ipsilateral OBs 34.42 ± 6.87 (mean ± SEM, Dunnett’s test,P ¼ 0.987 for contralateral vs. control and P ¼ 0.965 for ipsilateralvs. control; Fig. 8b). Therefore, DOPAC levels in female rats were notaffected by olfactory deprivation.The sex differences found in dopaminergic metabolites as a

response to olfactory deprivation affected the DOPAC ⁄ DA andHVA ⁄ DA ratios (Fig. 8c and d), indicative of intra- and extraneuronalmetabolism of DA. The lack of effect of olfactory deprivation onDOPAC contents in females, together with the reduction in DA levels,increased the DOPAC ⁄ DA ratio in the ipsilateral OB from deprivedfemale rats in comparison to that in deprived male OBs (1.34 ± 0.36in deprived females vs. 0.69 ± 0.12 in deprived males; mean ± SEM,Student’s t-test, P < 0.01; Fig. 8d). In males, there was a parallelreduction in DOPAC and DA content and hence a comparison of theDOPAC ⁄ DA ratio between the ipsilateral and control OBs of male rats

did not show significant differences. In contrast, the HVA ⁄ DA ratio inthe ipsilateral OB from male rats was increased in comparison to thatof the ipsilateral OB from female rats (2.24 ± 0.68 in deprived maleOBs vs. 1.17 ± 0.36 in deprived female OBs; mean ± SEM, Student’st-test, P < 0.01; Fig. 8c). As the decrease in DA concentration wassimilar in males and females, this result means that the decrease inHVA induced by olfactory deprivation, although not statisticallysignificant, tended to be higher in females than in males. The increasein the DOPAC ⁄ DA ratio in females and in the HVA ⁄ DA ratio in malesindicates an increase in DA turnover under olfactory deprivationconditions (Fig. 8c and d).

Discussion

The present results reveal sexual dimorphism in the dopaminergicsystem of the olfactory bulb of control and unilaterally deprived rats.Olfactory deprivation caused decreases in DA content as well as in thenumber of TH-IR cells in the ipsilateral OB. In addition, deprivationdifferentially affected males and females. The contralateral OBs didnot show any difference from the control OBs. It seems that intrinsicelements of the contralateral MOB were not directly affected byunilateral deprivation and, in this sense, similar results betweencontrol and contralateral MOBs have been reported in previous studies(Brunjes et al., 1985; Guthrie et al., 1990) and in the present paper.However, centrifugal systems are clearly affected bilaterally afterunilateral olfactory deprivation (Gomez et al., 2006, 2007), andbilateral naris occlusion leads to more dramatic changes in thecatecholaminergic system of the rat MOB than those reported forunilateral naris closure (Brinon et al., 2001), indicating a significantamount of information crossing between the hemispheres. These datatherefore indicate that the contralateral MOB cannot be considered andanalysed as a control without a detailed analysis.

Sexual dimorphism in control conditions

Sex differences regarding catechol contents in the OB are not theonly sign of sexual dimorphism that has been observed in thecatecholaminergic system (Vaccari et al., 1977; Simerly et al., 1985;

Fig. 7. Comparison between sexes of (a) the catechol contents (n ¼ 10, five males and five females) and of (b) the number of dopaminergic cells per unit area(n ¼ 8, four males and four females) in different layers of the MOB and in the AOB from ipsilateral OB of deprived animals. Results are shown as mean + SEM.*P < 0.05 between males and females.



Bhatt & Dluzen, 2005). These previous reports, together with ourresults, show that the catecholaminergic system is strongly dependenton sex, probably due to the regulation of TH gene activity by estrogen,a sex hormone (Thanky et al., 2002). Support for this hypothesis isthat TH mRNA levels in the MOB are also regulated by estrogen(Dluzen et al., 2002), and therefore events that alter estrogen levels(usually sexually dimorphic) can modulate OB catecholaminergicfunctions which, in turn, are involved in the detection and processingof olfactory stimuli (Dluzen et al., 2002).

As the OB was dissected out by cutting the olfactory peduncle, theMOB but also at least a part of the AOB were analysed by HPLC.Thus, sex differences in catechol contents may have been due tochanges in the MOB and ⁄ or AOB. However, this and previousimmunohistochemical analyses (Baker, 1986) have revealed that thenumber of catecholaminergic neurons in the AOB is almost negligible(< 1%) in comparison with the number of TH-IR cells in the MOB.Moreover, our results revealed sex differences in the number of TH-IRcells in the MOB but not in the AOB. Therefore, sex differences inDA, DOPAC and HVA contents must presumably be due todifferences in the number of dopaminergic neurons in the MOB. Thisis not the first time that sex differences have been demonstrated in themain olfactory system (Swann & Fibre, 1997; Weruaga et al., 2001;

Gu et al., 2003; Keverne, 2004) but it is the first time that the amountand the metabolism of a neurotransmitter has been shown to besexually dimorphic in the MOB. The dopaminergic system is not onlyfinely regulated by afferent stimulation but is also influenced by sex.As DA and its metabolites play an important role in the modulation ofolfactory transmission at the bulbar level, the dopaminergic systemcould be a suitable anatomical substrate for the reported sexdifferences in olfactory sensitivity, memory and discriminationproperties.Although the most abundant TH-IR cells in the MOB were located

in the GL, sex differences were found in the number of dopaminergiccells in the EPL. On average, EPL dopaminergic cells were larger thanthose in a juxtaglomerular position. Some authors have suggested thatthese cells as well as large cells in the GL would be tufted cells(Macrides & Davis, 1983; Baker, 1986; Gall et al., 1987). In thepresent study, we found both cells with the morphological features oftufted cells and cells whose aspect differed from that of tufted cells. Infact, PI staining shows tufted and mitral cells as large slightly stainedcells in comparison to the other bulbar cells, and our analysis revealedTH-IR cells without this PI-staining pattern. In addition, largepopulations of tufted (external, middle and inner) cells contain theneuropeptide CCK (Seroogy et al., 1985), although our analysis did

Fig. 8. Concentration of DA, DOPAC and HVA in control (white barrels), contralateral (grey barrels) and ipsilateral (black barrels) OBs in (a) males and in(b) females. Results shown as mean + SEM. **P < 0.01, ipsilateral vs. control OB. (c) HVA ⁄ DA and (d) DOPAC ⁄ DA ratios showing the different response ofmales and females under olfactory deprivation conditions. Results shown as means + SEM. **P < 0.01, males vs. females within the different OB groups.



not reveal a colocalization of CCK with TH in cells of the EPL or GL.Although some large TH-IR cells are tufted cells, it seems clear thatother dopaminergic cells are modulatory short-axon cells. It has beendemonstrated that EPL interneurons inhibit bulbar projection cells(mitral and tufted cells; Toida et al., 1996; Crespo et al., 2001;Hamilton et al., 2005) and that DA is also an inhibitory modulator ofmitral cells (Gall et al., 1987; Hsia et al., 1999). As TH-IR cells in theEPL seem to be interneurons and, based on the differences betweensexes in the number of dopaminergic cells and in DA contents, thedopaminergic inhibitory modulation from EPL cells to the bulbarprojection cells may be higher in the MOB of male rats than of femalerats, probably indicating a stronger lateral inhibition and thus a bettersignal discrimination in males than in females (Wilson & Sullivan,1995). This interpretation is well supported by recent studies whichhave demonstrated sex differences in olfactory sensitivity (Baum &Keverne, 2002; Pierman et al., 2006) and olfactory discriminationability (Wesson et al., 2006). Although behavioural interpretationsmust be carefully considered because of possible interspecies differ-ences, it is known that female mice show higher olfactory sensitivity(Baum & Keverne, 2002; Pierman et al., 2006) but lower olfactorydiscrimination than males (Wesson et al., 2006), which would agreewith the differences in the dopaminergic system observed in rats in thepresent study.The MOB continuously receives new cells through the rostral

migratory stream (Altman, 1969; Doetsch & Alvarez-Buylla, 1996).New cells are generated in the subventricular zone, from where theymigrate to the MOB through the rostral migratory stream and thenmature into neurochemically different cell subpopulations (Carletonet al., 2002). Colocalization BrdU-TH studies have reported that someof these newly generated cells differentiate into dopaminergic cells(Winner et al., 2006). Studies conducted over the past several yearshave identified steroid hormones (e.g. adrenal steroids, testosteroneand estrogen) and peptide hormones (such as prolactin) as potentialregulators of neurogenesis (Lennington et al., 2003). For example,adrenal steroids suppress whereas estrogen increases the production ofnew neurons in the hippocampus (Cameron & Gould, 1994; Tanapatet al., 1999), and prolactin stimulates the production of neuronalprogenitors in the subventricular zone (Shingo et al., 2003). Thenewly generated cells in adult animals differentiate mainly intogranule cells and periglomerular cells (Altman, 1969), and herestatistically significant sex differences were mainly observed in theEPL. However, because sex hormones regulate neurogenesis andhence the arrival of new neurons at the MOB, the differences observedin the number of dopaminergic cells between males and females couldbe due to differences between the sexes in the regulation ofneurogenesis (including proliferation, migration, differentiation andsurvival) in both developmental and adult stages.

Effects of unilateral olfactory deprivation

The reduction in DA content that occurred after 60 days of narisocclusion was correlated with a decrease not only in the number ofTH-IR juxtaglomerular cells found (in this and in previous studies;Philpot et al., 1998; Brinon et al., 2001) but also in the number ofTH-IR cells in the remaining layers, especially in the EPL. Peripheralafferent innervation is therefore required for the expression of the DAphenotype, not only in the GL but in the whole rodent MOB (Baker,1990; Baker et al., 1993).Sex differences in the number of TH-IR cells in the EPL from

control OBs coincided with sex differences in DA, DOPAC and HVAcontents. In addition, the similar numbers of dopaminergic cells in theEPL of deprived males and females agrees with similar bulbar DA and

DOPAC concentrations. It could thus be surmised that DA andDOPAC contents differed in control male and female animals becausethey had different numbers of TH-IR cells. In the same sense, DA andDOPAC levels would have been similar in deprived males and femalesbecause they had the same number of dopaminergic neurons in theEPL. Nevertheless, the reduction in DA and its metabolites was muchgreater than the decrease in the number of TH-IR cells; in fact, thedensity of TH-IR cells in female MOBs did not decrease afterdeprivation although that of DA did. DA as well as DOPAC and HVAcontent could vary among cells, and hence changes in content of DAand its metabolites cannot be explained only by differences in thenumber of TH-IR cells.Dopaminergic metabolism in the ipsilateral OB seems to be

different in males and females. DA can be metabolized by monoamineoxidase and catechol-O-methyltransferase to generate DOPAC andHVA (Wood & Altar, 1988). The HVA ⁄ DA (in males) andDOPAC ⁄ DA (in females) ratios, metabolic indices of DA activity,were more than doubled after deprivation as compared with controlconditions. The decrease in TH expression (Baker, 1990), togetherwith these results, seems to indicate that olfactory deprivation causes asimilar reduction in DA synthesis in both males and females, whereasthe degradation process of DA is sexually dimorphic. In fact, theHVA ⁄ DA or DOPAC ⁄ DA ratios were elevated after unilateralolfactory deprivation because HVA (in males) and DOPAC (infemales) levels were less diminished than those of DA. DOPACprimarily reflects the intraneuronal metabolism of DA, whereas HVAis a product of intra- and extraneuronal metabolism of released DA(Altar et al., 1984; Wood & Altar, 1988). The increase in theDOPAC ⁄ DA ratio in females points to a maintenance of theintraneuronal metabolism of DA (meaning that the intraneuronaldegradation of DA continues actively after olfactory deprivation,being independent of DA content, while extraneuronal degradationdecreases with the reduction in DA levels). This agrees with aprevious study reporting that olfactory nerve stimulation regulates DAcontent but does not change DOPAC concentrations (Philpot et al.,1998). The increase in the HVA ⁄ DA ratio and the decrease in DOPAClevels in male rats point, by contrast, to alterations in intraneuronaldopaminergic metabolism, whereas extraneuronal degradation seemsto be similar to that observed under control conditions. Taken together,these data indicate that not only DA synthesis but also DA degradationreacts to olfactory deprivation. However, alterations in dopaminergicdegradation, namely in dopaminergic metabolism, differ betweendeprived males and females, pointing to another sex difference in theresponse of the dopaminergic system in the deprived OB.The sex differences observed in the DOPAC ⁄ DA and HVA ⁄ DA

ratios are interesting because they suggest sex dissimilarities inmonoamine oxidase and ⁄ or catechol-O-methyltransferase enzymes,in DA transporter or in vesicular function, i.e. in the machineryrequired for DA release and ⁄ or degradation (Bhatt & Dluzen, 2005).Previous studies have shown sexual dimorphisms in the activity or inthe amount of these substances in other encephalic regions (Vaccariet al., 1977; Rivest et al., 1995; Bhatt & Dluzen, 2005). In fact, ithas recently been demonstrated that men have a markedly greaterDA release than women in the striatum (Munro et al., 2006). Inaddition, monoamine oxidase activity is higher in the whole brain ofadult female than of male rats (Skillen et al., 1961), and the samepattern has been observed in the human central nervous system(Robinson et al., 1971). It is known that both amount and activity ofDA transporter is greater in the female than in the male rodentstriatum (Rivest et al., 1995; Bhatt & Dluzen, 2005). All these datasuggest that intraneuronal dopaminergic metabolism is greater infemales than in males, which coincides with the sex differences



found in the DOPAC ⁄ DA and HVA ⁄ DA ratios in the OBs ofdeprived animals.

The present results suggest that the dopaminergic system modu-lates olfactory transmission differently in males and females.Moreover, in the OB, DA and its metabolites are regulated byafferent sensory activity and their responses to altered environmentalconditions also differ between the sexes. These differences suggestthat the modulation performed by DA on the olfactory signal isdependent on sex. Therefore, DA could be a key player in thedifferent olfactory sensitivities and discriminations shown by maleand female rats.

Acknowledgements

This work was supported by the Ministerio de Educacion y Ciencia (SAF2006–05705), Junta de Castilla y Leon and Fundacion MMA.

Abbreviations

AOB, accessory olfactory bulb; CCK, cholecystokinin; DA, dopamine;DOPAC, 3,4-dihydroxyphenylacetic acid; EPL, external plexiform layer; GL,glomerular layer; HPLC, high-performance liquid chromatography; HVA,homovanillic acid; IR, immunoreactive; MOB, main olfactory bulb; NC,neurocalcin; OB, olfactory bulb; PB, phosphate buffer, pH 7.4; PBS, phosphate-buffered saline; PI, propidium iodide; PV, parvalbumin; TH, tyrosine hydroxy-lase.

References

Altar, C.A., O’Neil, S. & Marshall, J.F. (1984) Sensorimotor impairment andelevated levels of dopamine metabolites in the neostriatum occur rapidlyafter intranigral injection of 6-hydroxydopamine or gamma-hydroxybutyratein awake rats. Neuropharmacology, 23, 309–318.

Altman, J. (1969) Autoradiographic and histological studies of postnatalneurogenesis. IV. Cell proliferation and migration in the anterior forebrain,with special reference to persisting neurogenesis in the olfactory bulb.J. Comp. Neurol., 137, 433–457.

Baker, H. (1986) Species differences in the distribution of substance P andtyrosine hydroxylase immunoreactivity in the olfactory bulb. J. Comp.Neurol., 252, 206–226.

Baker, H. (1990) Unilateral, neonatal olfactory deprivation alters tyrosinehydroxylase expression but not aromatic amino acid decarboxylase orGABA immunoreactivity. Neuroscience, 36, 761–771.

Baker, H., Morel, K., Stone, D.M. & Maruniak, J.A. (1993) Adult naris closureprofoundly reduces tyrosine hydroxylase expression in mouse olfactory bulb.Brain Res., 614, 109–116.

Baum, M.J. & Keverne, E.B. (2002) Sex difference in attraction thresholds forvolatile odors from male and estrous female mouse urine. Horm. Behav., 41,213–219.

Berkowicz, D.A. & Trombley, P.Q. (2000) Dopaminergic modulation at theolfactory nerve synapse. Brain Res., 855, 90–99.

Bhatt, S.D. & Dluzen, D.E. (2005) Dopamine transporter function differencesbetween male and female CD-1 mice. Brain Res., 1035, 188–195.

Brinon, J.G., Crespo, C., Weruaga, E., Martınez-Guijarro, F.J., Aijon, J. &Alonso, J.R. (2001) Bilateral olfactory deprivation reveals a selectivenoradrenergic regulatory input to the olfactory bulb. Neuroscience, 102,1–10.

Brunig, I., Sommer, M., Hatt, H. & Bormann, J. (1999) Dopamine receptorsubtypes modulate olfactory bulb gamma-aminobutyric acid type Areceptors. Proc. Natl Acad. Sci. USA, 96, 2456–2460.

Brunjes, P.C. (1994) Unilateral naris closure and olfactory system development.Brain Res. Rev., 19, 146–160.

Brunjes, P.C., Smith-Crafts, L.K. & McCarty, R. (1985) Unilateral odordeprivation: effects on the development of olfactory bulb catecholamines andbehaviour. Brain Res., 354, 1–6.

Cameron, H.A. & Gould, E. (1994) Adult neurogenesis is regulated by adrenalsteroids in the dentate gyrus. Neuroscience, 61, 203–209.

Carleton, A., Rochefort, C., Morante-Oria, J., Desmaisons, D., Vincent, J.D.,Gheusi, G. & Lledo, P.M. (2002) Making scents of olfactory neurogenesis.J. Physiol. (Paris), 96, 115–122.

Celio, M.R., Baier, W., Scharer, L., de Viragh, P.A. & Gerday, C. (1988)Monoclonal antibodies directed against the calcium binding proteinparvalbumin. Cell Calcium, 9, 81–86.

Crespo, C., Blasco-Ibanez, J.M., Marques-Mari, A.I. & Martınez-Guijarro, F.J.(2001) Parvalbumin-containing interneurons do not innervate granule cells inthe olfactory bulb. Neuroreport, 12, 2553–2556.

Davila, N.G., Blakemore, L.J. & Trombley, P.Q. (2003) Dopamine modulatessynaptic transmission between rat olfactory bulb neurons in culture.J. Neurophysiol., 90, 395–404.

Dluzen, D.E., Park, J.H. & Kim, K. (2002) Modulation of olfactory bulbtyrosine hydroxylase and catecholamine transporter mRNA by estrogen.Mol. Brain Res., 108, 121–128.

Doetsch, F. & Alvarez-Buylla, A. (1996) Network of tangential pathways forneuronal migration in adult mammalian brain. Proc. Natl Acad. Sci. USA, 93,14895–14900.

Ennis, M., Zhou, F.M., Ciombor, K.J., Aroniadou-Anderjaska, V., Hayar, A.,Borrelli, E., Zimmer, L.A., Margolis, F. & Shipley, M.T. (2001) DopamineD2 receptor-mediated presynaptic inhibition of olfactory nerve terminals.J. Neurophysiol., 86, 2986–2997.

Gall, C.M., Hendry, S.H., Seroogy, K.B., Jones, E.G. & Haycock, J.W. (1987)Evidence for coexistence of GABA and dopamine in neurons of the ratolfactory bulb. J. Comp. Neurol., 266, 307–318.

Gomez, C., Brinon, J.G., Barbado, M.V., Weruaga, E., Valero, J. & Alonso, J.R.(2005) Heterogeneous targeting of centrifugal inputs to the glomerular layerof the main olfactory bulb. J. Chem. Neuroanat., 29, 238–254.

Gomez, C., Brinon, J.G., Colado, M.I., Orio, L., Vidal, M., Barbado, M.V. &Alonso, J.R. (2006) Differential effects of unilateral olfactory deprivation onnoradrenergic and cholinergic systems in the main olfactory bulb of the rat.Neuroscience, 141, 2117–2128.

Gomez, C., Brinon, J.G., Orio, L., Colado, M.I., Lawrence, A.J., Zhou, F.C.,Vidal, M., Barbado, M.V. & Alonso, J.R. (2007) Changes in the serotonergicsystem in the main olfactory bulb of rats unilaterally deprived from birth toadulthood. J. Neurochem., 100, 924–938.

Gu, G., Cornea, A. & Simerly, R.B. (2003) Sexual differentiation of projectionsfrom the principal nucleus of the bed nuclei of the stria terminalis. J. Comp.Neurol., 460, 542–562.

Guillamon, A. & Segovia, S. (1997) Sex differences in the vomeronasalsystem. Brain Res. Bull., 44, 377–382.

Guthrie, K.M., Pullara, J.M., Marshall, J.F. & Leon, M. (1991) Olfactorydeprivation increases dopamine D2 receptor density in the rat olfactory bulb.Synapse, 8, 61–70.

Guthrie, K.M., Wilson, D.A. & y Leon, M. (1990) Early unilateral deprivationmodifies olfactory bulb function. J. Neurosci., 10, 3402–3412.

Halasz, N., Johansson, O., Hokfelt, T., Ljungdahl, A. & Goldstein, M. (1981)Immunohistochemical identification of two types of dopamine neuron in therat olfactory bulb as seen by serial sectioning. J. Neurocytol., 10, 251–259.

Hamilton, K.A., Heinbockel, T., Ennis, M., Szabo, G., Erdelyi, F. & Hayar, A.(2005) Properties of external plexiform layer interneurons in mouse olfactorybulb slices. Neuroscience, 133, 819–829.

Hsia, A.Y., Vincent, J.D. & Lledo, P.M. (1999) Dopamine depresses synapticinputs into the olfactory bulb. J. Neurophysiol., 82, 1082–1085.

Hsu, S.M., Raine, L. & Fanger, H. (1981) The use of antiavidin antibody andavidin-biotin-peroxidase complex in immunoperoxidase technics. Am. J.Clin. Pathol., 75, 816–821.

Keverne, E.B. (2004) Importance of olfactory and vomeronasal systems formale sexual function. Physiol. Behav., 83, 177–187.

Kosaka, K., Toida, K., Aika, Y. & Kosaka, T. (1998) How simple is theorganization of the olfactory glomerulus?: the heterogeneity of so-calledperiglomerular cells. Neurosci. Res., 30, 101–110.

Lennington, J.B., Yang, Z. & Conover, J.C. (2003) Neural stem cells and theregulation of adult neurogenesis. Reprod. Biol. Endocrinol., 1, 99.

Luo, M., Fee, M.S. & Katz, L.C. (2003) Encoding pheromonal signals in theaccessory olfactory bulb of behaving mice. Science, 299, 1196–1201.

Macrides, F. & Davis, B.J. (1983) The olfactory bulb. In Emson, P.C. (ed.),Chemical Neuroanatomy. Raven, New York, pp. 391–426.

Munro, C.A., McCaul, M.E., Wong, D.F., Oswald, L.M., Zhou, Y., Brasic, J.,Kuwabara, H., Kumar, A., Alexander, M.Ye, W. & Wand, G.S. (2006) Sexdifferences in striatal dopamine release in healthy adults. Biol. Psychiatry,59, 966–974.

Oberg, C., Larsson, M. & Backman, L. (2002) Differential sex effects inolfactory functioning: the role of verbal processing. J. Int. Neuropsychol.Soc., 8, 691–698.

Philpot, B.D., Men, D., McCarty, R. & Brunjes, P.C. (1998) Activity-dependentregulation of dopamine content in the olfactory bulbs of naris-occluded rats.Neuroscience, 85, 969–977.



Pierman, S., Douhard, Q., Balthazart, J., Baum, M.J. & Bakker, J. (2006)Attraction thresholds and sex discrimination of urinary odorants in male andfemale aromatase knockout (ArKO) mice. Horm. Behav., 49, 96–104.

Rivest, R., Falardeau, P. & Di Paolo, T. (1995) Brain dopamine transporter:gender differences and effect of chronic haloperidol. Brain Res., 692, 269–272.

Robinson, D.S., Davis, J.M., Nies, A., Ravaris, C.L. & Sylwester, D. (1971)Relation of sex and aging to monoamine oxidase activity of human brain,plasma, and platelets. Arch. Gen. Psychiatry, 24, 536–539.

Segovia, S. & Guillamon, A. (1993) Sexual dimorphism in the vomeronasalpathway and sex differences in reproductive behaviors. Brain Res. Rev., 18,51–74.

Seroogy, K.B., Brecha, N. & Gall, C. (1985) Distribution of cholecystokinin-like immunoreactivity in the rat main olfactory bulb. J. Comp. Neurol., 239,373–383.

Shingo, T., Gregg, C., Enwere, E., Fujikawa, H., Hassam, R., Geary, C., Cross,J.C. & Weiss, S. (2003) Pregnancy-stimulated neurogenesis in the adultfemale forebrain mediated by prolactin. Science, 299, 117–120.

Simerly, R.B., Swanson, L.W. & Gorski, R.A. (1985) The distribution ofmonoaminergic cells and fibers in a periventricular preoptic nucleus involvedin the control of gonadotropin release: immunohistochemical evidence for adopaminergic sexual dimorphism. Brain Res., 330, 55–64.

Skillen, R.G., Thienes, C.H. & Strain, L. (1961) Brain 5-hydroxytryptamine,5-hydroxytryptophan decarboxylase, and monoamine oxidase of normal,thyroid-fed, and propylthiouracil-fed male and female rats. Endocrinology,69, 1099–1102.

Stone, D.M., Grillo, M., Margolis, F.L., Joh, T.H. & Baker, H. (1991)Differential effect of functional olfactory bulb deafferentation on tyrosinehydroxylase and glutamic acid decarboxylase messenger RNA levels inrodent juxtaglomerular neurons. J. Comp. Neurol., 311, 223–233.

Swann, J. & Fiber, J.M. (1997) Sex differences in function of a pheromonallystimulated pathway: role of steroids and the main olfactory system. BrainRes. Bull., 44, 409–413.

Tanapat, P., Hastings, N.B., Reeves, A.J. & Gould, E. (1999) Estrogenstimulates a transient increase in the number of new neurons in the dentategyrus of the adult female rat. J. Neurosci., 19, 5792–5801.

Thanky, N.R., Son, J.H. & Herbison, A.E. (2002) Sex differences in theregulation of tyrosine hydroxylase gene transcription by estrogen in thelocus coeruleus of TH9-LacZ transgenic mice. Mol. Brain Res., 104, 220–226.

Toida, K., Kosaka, K., Heizmann, C.W. & Kosaka, T. (1996) Electronmicroscopic serial sectioning ⁄ reconstruction study of parvalbumin-contain-ing neurons in the external plexiform layer of the rat olfactory bulb.Neuroscience, 72, 449–466.

Vaccari, A. (1980) Sexual differentiation of monoamine neurotransmitters. InParvez, H. & Parvez, S. (eds), Biogenic Amines in Development. Elsevier,Amsterdam, pp. 327–351.

Vaccari, A., Brotman, S., Cimino, J. & Timiras, P.S. (1977) Sex differentiationof neurotransmitter enzymes in central and peripheral nervous systems.Brain Res., 132, 176–185.

Weruaga, E., Brinon, J.G., Porteros, A., Arevalo, R., Aijon, J. & Alonso, J.R.(2001) A sexually dimorphic group of atypical glomeruli in the mouseolfactory bulb. Chem. Senses, 26, 7–15.

Wesson, D.W., Keller, M., Douhard, Q., Baum, M.J. & Bakker, J. (2006)Enhanced urinary odor discrimination in female aromatase knockout (ArKO)mice. Horm. Behav., 49, 580–586.

Wilson, D.A. & Sullivan, R.M. (1995) The D2 antagonist spiperone mimics theeffects of olfactory deprivation on mitral ⁄ tufted cell odor response patterns.J. Neurosci., 15, 5574–5581.

Winner, B., Geyer, M., Couillard-Despres, S., Aigner, R., Bogdahn, U., Aigner,L., Kuhn, G. & Winkler, J. (2006) Striatal deafferentation increasesdopaminergic neurogenesis in the adult olfactory bulb. Exp. Neurol., 197,113–121.

Wood, P.L. & Altar, C.A. (1988) Dopamine release in vivo from nigrostriatal,mesolimbic, and mesocortical neurons: utility of 3-methoxytyraminemeasurements. Pharmacol. Rev., 40, 163–187.



Documents

Sex differences in catechol contents in the olfactory bulb of ...Sex differences in catechol contents in the olfactory bulb of control and unilaterally deprived rats C. Go´mez,1 J