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Psychoneuroendocrinology (2008) 33, 437–445

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1These authors co

Characterization of central and peripheralcomponents of the hypothalamus–pituitary–adrenalaxis in the inbred Roman rat strains

Javier Carrascoa,1, Cristina Marqueza,1, Roser Nadalb, Adolfo Tobenac,Albert Fernandez-Teruelc, Antonio Armarioa,�

aUnitat de Fisiologia Animal (Facultat de Biociencies), Institut de Neurociencies,Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, SpainbUnitat de Psicobiologia (Facultat de Psicologia), Institut de Neurociencies,Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, SpaincUnitat de Psicologia Medica (Facultat de Medicina), Institut de Neurociencies,Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

Received 28 September 2007; received in revised form 27 December 2007; accepted 3 January 2008

KEYWORDSCRF;Glucocorticoidreceptors;Mineralocorticodreceptors;Central amygdala;Bed nucleus striaterminalis;Inbred Roman ratstrains

nt matter & 2008uen.2008.01.001

thor. Tel.: +34 93ntonio.armario@u

ntributed equally

SummarySeveral studies performed in outbred Roman high- and low-avoidance lines (RHA and RLA,respectively) have demonstrated that the more anxious line (RLA) is characterized by ahigher hypothalamic–pituitary–adrenal (HPA) response to certain stressors than the lessanxious one (RHA). However, inconsistent results have also been reported. Takingadvantage of the generation of an inbred colony of RLA and RHA rats (RHA-I and RLA-I,respectively), we have characterized in the two strains not only resting and stress levels ofperipheral HPA hormones but also central components of the HPA axis, including CRF geneexpression in extra-hypothalamic areas. Whereas resting levels of ACTH and corticosteronedid not differ between the strains, a greater response to a novel environment was found inRLA-I as compared to RHA-I rats. RLA-I rats showed enhanced CRF gene expression in theparaventricular nucleus (PVN) of the hypothalamus, with normal arginin-vasopressin geneexpression in both parvocellular and magnocellular regions of the PVN. This enhanced CRFgene expression is not apparently related to altered negative corticosteroid feedback assimilar levels of expression of brain glucorticoid and mineralocorticoid receptors werefound in the two rat strains. CRF gene expression tended to be higher in the centralamygdala and it was significantly higher in the dorsal region of the bed nucleus of striaterminalis (BNST) of RLA-I rats, while no differences appeared in the ventral region ofBNST. Considering the involvement of CRF and the BNST in anxiety and stress-related

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to this paper.

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behavioral alterations, the present data suggest that the CRF system may be a criticalneurobiological substrate underlying differences between the two rat strains.& 2008 Elsevier Ltd. All rights reserved.

1. Introduction

The outbred Roman high- and low-avoidance lines (RHA andRLA, respectively) were initially obtained by geneticselection on the basis of learning performance in a two-way avoidance task in a shuttle box (Bignami, 1965;Broadhurst and Bignami, 1965). Most of the studieshave been done using the rat stocks bred in Switzerland(RHA/Verh and RLA/Verh; for reviews, see Driscoll andBattig, 1982; Escorihuela et al., 1995; Fernandez-Teruelet al., 1997, 2002b; Driscoll et al., 1998), as well as withsub-stocks generated in different labs (e.g. Castanon et al.,1992, 1994, 1995; Steimer and Driscoll, 2003; Giorgi et al.,2007). It was soon realized that the extreme differences inactive avoidance between the two lines were mainly due todifferences in emotionality/fearfulness rather than inlearning capabilities, RLA rats being more emotional/fearfulthan RHA rats in most of the (typical) unconditioned- andconditioned-anxiety tasks in which they have been tested todate (for reviews, see Driscoll and Battig, 1982; Ferre et al.,1995; Fernandez-Teruel et al., 1997, 2002b; Driscoll et al.,1998; Escorihuela et al., 1999; Steimer and Driscoll, 2003,2005) and displaying more passive (freezing) strategies whenfacing highly stressful situations (e.g. Driscoll et al., 1980;Ferre et al., 1995; Steimer and Driscoll, 2003, 2005; Aguilaret al., 2004). Thus, RLA rats are considered as a good animalgenetic model of anxiety/fearfulness. In fact, anxiolyticsare effective in RLA but not in RHA rats (Martin et al., 1982;Fernandez-Teruel et al., 1991; Corda et al., 1998; Steimerand Driscoll, 2003; Torres et al., 2007), which also appear tohave a higher brain GABAergic tone (Giorgi et al., 1994;Bentareha et al., 1998). Interestingly, there is evidence thatother behavioral aspects, in addition to anxiety/fearfulness,have been selected in parallel with active avoidance, as RHArats are characterized as more novelty/substance-seekingsubjects (e.g. Fernandez-Teruel et al., 2002a, 1992, 1997;Razafimanalina et al., 1996; Siegel, 1997; Driscoll et al.,1998; Escorihuela et al., 1999; Giorgi et al., 2007).

The characterization of hypothalamic–pituitary–adrenal(HPA) function in the Roman rat lines has generatedcontroversial results. In the first description of the putativehormonal differences between the two lines, Gentsch et al.(1982) reported similar resting levels of stress hormones(ACTH, corticosterone and prolactin), but a greaterresponse of the three hormones to mild stressors (i.p.injection or exposure to novel environments) in RLA ascompared to RHA. Interestingly, the differences were notobserved with more severe stressors such as ether, im-mobilization or footshock (Gentsch et al., 1982). Onepotential drawback of this interesting study is that hormonalmeasurement was done only at 10min after initial exposureto the stressful situations, which is an appropriate time forACTH but not for corticosterone (Le Mevel et al., 1979). Inspite of this initial consistency, further studies have notobtained a picture so clear. Thus, Walker et al. (1989)

reported, using a similar time-course as the previousauthors, higher resting plasma levels of ACTH (but notcorticosterone) in RLA rats, with no differences betweenlines in the ACTH response to an open field or ether, inabsolute terms. In these experiments, corticosteroneresponse was lower in RHA than RLA rats after exposure tothe open field but not to ether. In contrast, in a sub-stockmaintained in Bordeaux, Castanon et al. (1992) did not finddifferences between the two lines either in basal or in stress(novel environments) levels of ACTH and corticosterone,whereas prolactin response was higher in RLA rats. Thisapparent discrepancy may be due to the age of the rats sincedifferences between the two lines regarding HPA respon-siveness appear to emerge between the 14th and the 20thweek (Castanon et al., 1994) and the consistently higher HPAresponse to the novel environment has always beenobserved in rats 5 months old or older (Gentsch et al.,1982; Aubry et al., 1995; Fernandez-Teruel et al., 2002b).Whether this is an effect of age per se or the result ofaccumulative environmental influences is unclear (Castanonet al., 1995).

There is only limited information concerning the putativemechanisms involved in the differential HPA response tomild stressors in RHA–RLA rats and the results are puzzling.Higher plasma ACTH levels after exogenous corticotropin-releasing factor (CRF) administration have been reported inRLA as compared to RHA rats (Walker et al., 1989), an effectthat was associated with a higher in vitro ACTH release bydispersed pituitary cells in culture. These changes were notdue to an altered pituitary ACTH content. These datasuggest an increased capability of corticotropes from RLArats to respond to endogenous CRF release, which maycontribute to the enhanced stress-induced ACTH release.Interestingly, a lower apparent number of glucocorticoidreceptors (GR) were detected in the pituitary of RLA rats,whereas in the hippocampus a normal number of GR but alower number of mineralocorticoid receptors (MR) werefound (Walker et al., 1989). Since both GR and MR areinvolved in the control of the HPA response to stress (Ratkaet al., 1989; Spencer et al., 1998), the data are suggestiveof impaired negative glucocorticoid feedback at the level ofthe hippocampus and the pituitary. At the level of theparaventricular nucleus (PVN), normal expression of CRF butenhanced expression of vasopressin in parvocellular PVNneurons has been reported in the same study in which RLArats showed an enhanced corticosterone response to theopen field (Aubry et al., 1995). Since enhanced expression ofvasopressin in parvocellular PVN neurons is observed underconditions of chronically enhanced activity of the HPA axis(Aguilera and Rabadan-Diehl, 2000), these results givesupport to the hypothesis of central HPA hyperactivity inRLA rats.

CRF plays a pivotal role not only in the control of the HPAaxis, but also in a wide range of physiological and behavioralresponses to stress (Dunn and Berridge, 1990; Koob, 1999)

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through populations of CRF neurons located in the PVNitself, and also in several brain areas such as the centralamygdala (CeA), the dorsal and ventral bed nucleus of striaterminalis (BNST) and the Barrington nucleus. At present,little is known about differences between the Roman lines inbrain CRF populations, but it has been recently reported inthe inbred Roman rats that RLA showed a higher number ofCRF+ neurons in the CeA than RHA rats (Yilmazer-Hanke etal., 2002), which may contribute to explain the enhancedemotionality of RLA rats.

A process of inbreeding of the Roman outbred lines(RHA/Verh and RHA/Verh) has been carried out since 1993(Escorihuela et al., 1997, 1999; Driscoll et al., 1998;Fernandez-Teruel et al., 2002b), starting in Zurich (ETH-Z;Dr. P. Driscoll) and being continued at the AutonomousUniversity of Barcelona (UAB; A. Fernandez-Teruel, R.M.Escorihuela and A. Tobena), leading to the RHA-I and RLA-Iinbred strains. In general, behavioral differences betweenthe two strains, including two-way active avoidance in theshuttle-box, have been maintained during the process(Escorihuela et al., 1997, 1999; Fernandez-Teruel et al.,2002b; Aguilar et al., 2004), but hormonal studies have beenscarce (Steimer et al., 1997). Therefore, the aim of thepresent work was to characterize HPA axis reactivity in theseinbred lines. In addition, vasopressin gene expression wasstudied not only in the parvocellular, but also in themagnocellular region of the PVN as higher vasopressin geneexpression has been reported in high-anxiety behavior (HAB)as compared to low-anxiety behavior (LAB) rats, which havebeen genetically selected for extreme differences in anxietyusing a clearly distinct criterion, the time spent in the openarms of the elevated plus-maze (Keck et al., 2002; Landgrafand Wigger, 2003).

2. Material and methods

2.1. Animals and general procedure

Twenty-five-month-old male rats (RHA-I n ¼ 10; RLA-I n ¼10; from the 28th inbreeding generation) bred in the animalfacilities of the Medical Psychology Unit (UAB) were used inthis experiment. They were housed two per cage in standardconditions of temperature (22 1C71) and on a 12–12light–dark schedule (lights on at 0800 h). Food and waterwere provided ad libitum. Cages were cleaned once a week.The experimental protocol was approved by the committeeof Ethics of the Universitat Autonoma de Barcelona and wascarried out in accordance with the European CommunitiesCouncil Directive (86/609/EEC).

The experimental procedures were all done in themorning. Blood samples were taken by tail-nick procedurein resting conditions, in order to evaluate basal hormonelevels and to habituate the animals to the samplingprocedure. One week later, all the animals were exposedto a square novel environment for 20min. A 20-min exposureto the novel environment was chosen as optimum, becausemaximum plasma corticosterone levels are never achievedbefore 20min and the ACTH response starts declining lateron. The two rats of the same cage were simultaneouslyexposed to two similar novel environments to avoid cohortremoval effects. Blood samples were taken by tail-nick just

immediately after the exposure to the novel environment,and at 30 and 60min post-novel environment. The two post-novel environment sampling periods were added to obtain abetter picture of the hormonal response. Basal sampleswere not taken prior to the novel environment in orderto prevent possible interferences with behavior andhormonal response. The animals were returned to theirhome cages and the animal room after each bloodsampling.

The tail-nick consisted of gently wrapping the animalswith a cloth, making a 2mm incision at the end of one of thetail arteries and then massaging the tail while collecting,within 2min, 300 ml of blood into ice-cold EDTA capillarytubes (Sarsted, Granollers, Spain). Within the same day,repeated samples were obtained, making only one incision.Plasma obtained after centrifugation was stored at �30 1Cuntil assay.

One week after OF exposure, animals were sacrificedunder resting conditions and adrenal glands were extractedand weighed. Animals were anesthetized with 80mg/kgketamine (Merial Laboratories, Barcelona, Spain) and10mg/kg xylazine (Bayer, Barcelona, Spain) and perfusedvia the ascending aorta with ice-cold 0.9% saline for 2min,followed by 4% paraformaldehyde (PFA) in DEPC-treatedpotassium phosphate-buffered saline (KPBS) for 10min.After perfusion, brains were removed from the skull,postfixed overnight in the same fixative and immersed in30% sucrose solution in KPBS at 4 1C for cryoprotection untilthey sank. At that point, brains were frozen and stored at�80 1C until further processing.

2.2. Apparatus

Dark square plastic boxes (40� 40� 40 cm3) located in aquite room were used as novel environments. A 40W whitebulb was placed 1.20m above the center of the apparatus.Each animal was placed initially in the periphery of theapparatus facing the wall. Blood sampling procedure wasdone in a separate room.

2.3. Biochemical analysis

Plasma ACTH and corticosterone levels were determined bydouble-antibody radioimmunoassay (RIA) procedures cur-rently used in our laboratory. ACTH RIA used 125I-ACTH(Amersham, Spain) as the tracer, rat synthetic ACTH 1-39(Sigma, Spain) as the standard and an antibody raisedagainst rat ACTH (rb 7) kindly provided by Dr. W.C. Engeland(Department of Surgery, University of Minnesota, Minneapo-lis, USA). Corticosterone RIA used 125I-carboximethyloxime-tyrosine-methyl ester (ICN-Biolink 2000, Spain) as the tracer,synthetic corticosterone (Sigma) as the standard and anantibody raised in rabbit against corticosterone-carboxi-methyloxime-BSA kindly provided by Dr. G. Makara (Instituteof Experimental Medicine, Budapest, Hungary). We followedthe RIA protocol recommended by Dr. G. Makara (plasmacorticosteroid-binding globulin was inactivated by low pH)(Bagdy and Makara, 1994). All samples to be compared wererun in the same assay to avoid inter-assay variability. Theintra-assay coefficient of variation was o8% for bothhormones.

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2.4. In situ hybridization

2.4.1. Probe preparationThe CRF probe was generated from a pGEM-4Z plasmidcontaining an EcoRI fragment (1.2 kb) of cDNA of the rat(Dr. K. Mayo, Northwestern University, Evanston, IL, USA),linearized with HindIII. The arginin-vasopressin (AVP) probewas generated from a pSp 65 plasmid containing an SMAI/PSTI fragment (230 bp) of cDNA of the rat (Dr. Richter,Institute of Cell Biology and Institute of Clinical Neurobiol-ogy, University of Hamburg, Hamburg, Germany), linearizedwith HindIII. The GR probe was transcribed from a 500-bprat cDNA fragment that encodes for the N-terminal region ofthe rat liver GR (courtesy of Dr. K.R. Yamamoto and Dr. R.Miesfeld, Department of Biochemistry, University of Arizona,Tucson, AZ, USA). This fragment was subcloned from a 2.8-kb fragment and transfected into pGEM3 plasmid (courtesyof Dr. M.C. Bohn, Department of Pediatrics, NorthwesternUniversity Medical School, Chicago, IL, USA). It waslinearized with BamHI. MR probe was generated from apBS SK+ plasmid containing an EcoRI/HindIII fragment(550 bp) from a 30 coding region and 30 untranslated regionof rat MR cDNA (courtesy of Dr. K.R. Yamamoto and Dr. R.Miesfeld, Department of Biochemistry, University of Arizona,Tucson, AZ, USA). It was linearized with EcoRI. Radioactiveantisense cRNA copies were generated using a transcriptionkit (Roche, Germany) in the presence of [a-35S]-UTP (specificactivity 41000 Ci/mmol, GE Healthcare, UK). The cRNAwere precipitated with the ammonium acetate method,resuspended in 10mM Tris/1mM EDTA, pH 8.0, and storedat �20 1C.

2.4.2. In situ hybridization histochemistryThe protocol used was adapted from Simmons et al. (1989).All solutions were pretreated with DEPC and sterilizedbefore use. Sections were post-fixed in 4% PFA+Borax,digested with 0.01mg/ml proteinase K (Roche, Germany),acetylated in 0.25% acetic anhydride, washed in 2� SCC,dehydrated through graded concentrations of ethanol andair-dried. Thereafter, 100 ml of hybridization buffer contain-ing 1� 106 dpm of the labelled probe was spotted onto eachslide and sealed with a coverslip. After a 16–18 h incubationin a humid chamber at 60 1C, the slides were washed indescending concentrations of SSC containing 1mM DTT(Sigma, Spain), including one wash at 60 1C, digested withRNase A (0.02mg/ml, GE Healthcare, UK), dehydratedthrough a series of ethanol solutions and air-dried. Theslides were then exposed to an autoradiography film (XAR-5Kodak Biomax MR, Kodak, Spain). In case of AVP, afterdeveloping films, the slides were cleaned and defatted witha series of ethanol and xylene and dipped into LM-1 emulsion(GE Healthcare, UK) and developed after 24 h. The slideswere then counterstained with 0.25% thyonin (Sigma,Spain), dehydrated through increasing concentrations ofethanol, cleared in xylene and coverslipped with DPX(Electron Microscopy Sciences, PA, USA).

2.4.3. Image analysisFor CRF, GR and MR mRNAs densitometric analyses weredone on the autoradiography films. The mRNA levels weresemiquantitatively determined in three sections per brain

area (both hemispheria pooled) and animal. The sections tobe analyzed were photographed under a 4� microscopeobjective (Eclipse E400, Nikon, Japan) with DXM1200 digitalcamera (Nikon, Japan) and subsequently quantified withScion Image software (W. Rasband, NIH, USA; available onthe web at http://rsb.info.nih.gov/nih-image or http://www.scioncorp.com). The optimal detection threshold wasset for every brain area under analysis, in order to select thearea of interest without detecting any background signal.Measures were obtained in arbitrary units (square pix-els� average sum gray). In all cases, the intensity of thesignal was within the linear range as evaluated by the 14Cmicroscales (GE Healthcare, UK). For AVP mRNA, the lateralmagnocellular subdivision of the PVN was analyzed asdescribed above and the medial parvocellular subdivisionof the PVN was analyzed on the microautoradiograms (LM-1emulsion-covered sections). Developed sections were coun-terstained with thyonin. A cluster of silver grains associatedwith a single cell was measured by placing individuallabelled cells within a circle of fixed diameter. AVP-labelledmagnocellular neurons were excluded from the analysis onthe basis of size and relative grain density. Background graindensities were estimated from 20 cells per section per rat inthe PVN of the thalamus. Cells were considered specificallylabelled if the grain density was a minimum of five times thebackground (Kovacs and Sawchenko, 1996).

2.5. Statistical analysis

The statistical analysis was performed using the ‘‘StatisticalPackage for Social Science’’ (SPSS, Version 13). Normalityof the data was evaluated by means of the Kolmogorov–

Smirnov test. To compare differences between strainsregarding resting hormone levels and in gene expression,t-tests were used. To analyze ACTH and corticosteronechanges in relation to the strain, a repeated-measuresMANOVA was used, ‘‘Strain’’ being (RHA-I or RLA-I) thebetween-subject’s factor and ‘‘time’’ (resting levels, andlevels immediately after the novel environment and 30 and60min post-novel environment) the within-subject’s factor.Resting levels which were obtained on a different day andfound not to differ between strains were also included in theANOVA. Significance was set at po0.05.

3. Results

No differences either in body weight or in absolute andrelative adrenal weights were observed between the strains(Table 1). Similarly, direct comparison of resting ACTH andcorticosterone levels with t-test revealed no differencesbetween strains. The MANOVA revealed greater ACTH andcorticosterone responses to the novel environment in RLA-Ias compared to RHA-I rats. For ACTH: significant effectsof sampling time (F(3,54) ¼ 123.8, po0.001), strain(F(1,18) ¼ 30.8, po0.001) and the interaction samplingtime by strain (F(3,54) ¼ 8.9, po0.001) were found.Further analysis of the interaction revealed that RLA-Ianimals showed a greater ACTH response immediately afterstress (F(1,19) ¼ 34.7, po0.001) and during both post-stressperiods (post 30min: F(1,19) ¼ 25.0, po0.001; post 60min:F(1,19) ¼ 9.3, po0.01), but no differences were found

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Figure 1 Effects of 20-min exposure of RHA-I and RLA-I rats toa novel environment on plasma ACTH (panel A) and corticoster-one (panel B) levels. Means and SEM (n ¼ 10 for each strain) arerepresented. For ACTH, comparisons between strains at thesame sampling time are as follows: *po0.05; **po0.01;***po0.001. For corticosterone, global differences betweenstrains were found (*p ¼ 0.014). Differences with respect tobasal levels are not indicated (see text).

Figure 2 CRF (A) and AVP (B) gene expression in the PVN ofRHA-I and RLA-I rats sacrificed under resting conditions. Resultsare shown as mean7SEM (n ¼ 10 for each strain). CRF mRNAlevels (corresponding to the parvicellular PVN: pPVN) and AVPmRNA levels corresponding to the magnocellular PVN (mPVN)are expressed in arbitrary units (AU). In the pPVN, the numberof neurons expressing AVP (in cells/mm2) are indicated.Representative sections are shown above the graphics;*p ¼ 0.019.

Table 1 Comparison of adrenals weights between RHAand RLA rats.

Body weight(BW, g)

Absoluteadrenalweight (mg)

Relativeadrenalweight (mg/100 g BW)

RHA 426.078.7 51.770.9 12.270.4RLA 456.6717.6 50.371.0 11.270.5

Means7SEM (n ¼ 10) are represented. No significant differ-ences between the two lines were observed.

Figure 3 CRF mRNA levels determined in the central amygdala(CeA) and the dorsal and ventral aspects of bed nucleus of striaterminalis (BNST) of RHA-I and RLA-I rats sacrificed underresting conditions. Mean7SEM (n ¼ 10 for each strain) arerepresented in arbitrary units (AU). Representative sections areshown above the graphics; **p ¼ 0.007.

Hypothalamus–pituitary–adrenal axis in Roman lines 441

under resting (basal) conditions (Figure 1A). Moreover, RLA-Ianimals showed higher corticosterone levels than RHA-Iindependently of the sample time: significant effect oftime (F(3,54) ¼ 49.9, po0.001) and strain (F(1,18) ¼ 7.4,p ¼ 0.014), but not significant interaction time by strain(Figure 1B).

The same animals were sacrificed under basal conditionsseveral days after stress to study central aspects of the HPAaxis. RLA-I rats showed enhanced CRF gene expression in thePVN (t(11.9) ¼ 2.7, p ¼ 0.019), whereas no between-straindifferences were found in AVP mRNA levels in the magno-cellular PVN or in the number of parvocellular neuronsexpressing vasopressin (Figure 2A and B). In the extended

amygdala regions studied (Figure 3), differences betweenstrains regarding CRF gene expression were only marginallysignificant in the CeA (t(17) ¼ 1.8, p ¼ 0.083), higher in thedorsal aspects of the BNST in RLA-I as compared with RHA-Irats (t(18) ¼ 3.1, p ¼ 0.007) and similar in the ventralregion of the BNST.

GR gene expression was similar in the two lines in thedifferent regions of the hippocampal formation measured(DG, CA1) as well as in the mPFC and PVN (Figure 4). Nodifferences in MR expression were found in the hippocampalformation (Figure 4).

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Figure 4 MR and GR mRNA levels determined in several brainregions of RHA-I and RLA-I rats sacrificed under restingconditions. These regions were medial prefrontal cortex(mPFC), CA1, dentate gyrus (DG) and PVN for GR, and CA1and DG for MR. Mean7SEM (n ¼ 10 for each strain) arerepresented in arbitrary units (AU).

J. Carrasco et al.442

4. Discussion

The present results confirm that the greater HPA response toa mild stressor (exposure to a novel environment) of RLA ascompared to RHA rats, very often reported in outbredRoman lines, has been maintained after the inbreedingprocess. Resting levels of HPA hormones were similar in thetwo strains. This enhanced HPA response to stress in RLA-Irats was associated with an enhanced CRF gene expressionin the PVN and the dorsal BNST.

Although some divergent results, either among differentlaboratories or between the Roman outbred lines vs. inbredstrains, have been reported concerning between-line/straindifferences in the hyponeophagia test (Ferre et al., 1995;Steimer et al., 1998; Escorihuela et al., 1999) and in theelevated plus-maze (% time spent in the open arms;Escorihuela et al., 1999; Yilmazer-Hanke et al., 2002;Steimer and Driscoll, 2003), it should be noted that theinbred Roman strains, derived from the outbred RHA/Verhand RLA/Verh stocks, appear to have retained most of thecharacteristic differences described between the outbredlines: for instance, much better performance of RHA-I rats inthe two-way active avoidance task, increased exploratoryactivity and reduced self-grooming in several different novelenvironments, often including open/illuminated sections(open-field, holeboard, elevated plus-maze, light–dark box,light–dark tunnel maze, elevated ‘zero’ maze—unpub-lished), and enhanced exploration of holes and novel objectsin the holeboard (Escorihuela et al., 1997, 1999; Fernandez-Teruel et al., 2002b). Conversely, and also in accordancewith their higher emotionality/fearfulness, RLA-I rats showenhanced baseline, stress-potentiated and fear(cue)-poten-tiated startle response (Schwegler et al., 1997; Corda et al.,1998; Aguilar et al., 2000; Yilmazer-Hanke et al., 2002;Lopez-Aumatell et al., 2005), as well as higher context andcue-conditioned freezing and shock-induced suppression ofdrinking (Lopez-Aumatell et al., 2005).

We observed no differences between the two inbredstrains in body weight, absolute adrenal weight or relativeadrenal weight. In general, results regarding body weight

are not consistent (Driscoll and Battig, 1982; Walker et al.,1989; Meerlo et al., 1997), but greater relative adrenalweight has been consistently reported in RHA vs. RLA rats(e.g. Gentsch et al., 1981), which may not be apparent inyoung (o4 months) animals (Castanon et al., 1994). Thepresent data suggest that adrenal weight is likely to beunrelated to the main behavioral characteristics of RHA–

RLA, as well as with the reported differences in central andpituitary HPA function. This conclusion is supported by thefinding that Syracuse high-avoidance rats showed loweradrenal weight than Syracuse low-avoidance rats, despite acriterion of genetic selection similar to RHA–RLA rats thatalso resulted in higher anxiety in the Syracuse low-avoidance animals (Brush, 2003). In accordance with thelack of differences in adrenal weight between RHA-I andRLA-I rats, basal levels of ACTH and corticosterone weresimilar in both lines. In contrast, a markedly enhancedresponse of the two hormones to the novel environment wasfound in RLA-I as compared to RHA-I. These data confirmprevious results also obtained in the inbred strains (Steimeret al., 1997), suggesting that the differences between thelines concerning the HPA axis may be even more consistentamong labs after the inbreeding process.

The study of central gene expression of several majorcomponents of the HPA axis only revealed differences in CRFgene expression in the PVN, RLA-I rats showing increasedmRNA levels suggestive of an enhanced CRF activity. To ourknowledge, this is the first report demonstrating that thiskey central regulator of the HPA axis is altered between theRoman lines/strains. Previous reports with the outbred linesfailed to find differences (Aubry et al., 1995). Enhanced CRFgene expression closely follows enhanced CRF release intothe median eminence and, therefore, these data arecompatible with the increased HPA responsiveness duringexposure to a novel environment observed in RLA-I rats. Incontrast, the present data did not reveal any difference inAVP gene expression either in the magnocellular region ofthe PVN or in parvocellular neurons. Since there arescattered magnocellular AVP+ neurons in the parvocellularregions, we counted the number of AVP+ neurons in thefunction of grain density around the cells (a threshold of5-fold the background) and no between-strain differenceswere observed. These data contrast with those of Aubryet al. (1995), who reported a higher density of radioactivegrains in parvocellular AVP neurons of outbred RLA/Verhrats. When density of grains per cell in addition to thenumber of AVP+ cells were measured in the presentexperiment, we confirmed the lack of between-linesdifferences in AVP expression. We have no clear explanationfor these discrepancies, but there are at least threepossibilities. First, inbred strains may somewhat differ fromtheir outbred counterparts. Second, environmental factorsassociated to husbandry may differ between laboratories,and this would differentially affect parvocellular AVPexpression that is usually increased under conditions ofchronic stress (Aguilera and Rabadan-Diehl, 2000; Armario,2006). Third, Aubry et al. analyzed AVP gene expression 4 hafter exposure to a large and presumably highly stressfulopen field so that RLA as compared to RHA rats may becharacterized by a greater stress-induced AVP gene expres-sion rather than by differences in resting conditions.Interestingly, at our laboratory, HAB rats have also been

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Hypothalamus–pituitary–adrenal axis in Roman lines 443

characterized by increased CRF mRNA levels in the PVN(Marquez et al., in preparation), suggesting that both pairsof lines have in common enhanced CRF gene expression inthe PVN.

Whereas CRF gene expression in the PVN was greater inRLA-I than in RHA-I rats, no differences were found in theCeA. We analyzed CRF gene expression in these areas,because there is evidence that it may be related to stressfuland anxiety reactions (Koob, 1999). In addition, it has beenrecently reported that RLA-I rats have a higher number ofCRF+ neurons in the CeA (but not other amygdala nuclei)than RHA-I rats (Yilmazer-Hanke et al., 2002). Interestingly,in the present experiment CRF gene expression in the CeAwas marginally higher in RLA-I as compared to RHA-I rats.Relative discrepancies between the two studies may beexplained by the fact that between-strain differences in thenumber of CRF+ neurons may be compensated by enhancedCRF gene expression. Alternatively, the inbreeding processmay have reduced the differences in the population of CeACRF neurons between both strains. In any case, dissociationbetween CRF gene expression in the PVN and the CeA is notsurprising as CRF gene expression in those areas isindependent of each other (e.g. Watts and Sanchez-Watts,1995).

We also studied CRF gene expression in two regions of theBNST, an area intimately related to the amygdala. Whereas ahigher CRF gene expression was found in the dorsal region ofthe BNST of RLA-I as compared to RHA-I rats, no differenceswere observed in the ventral BNST. CRF gene expression inthese two regions has been found to be differentiallysensitive to emotional stressors and pharmacological manip-ulations (Shalev et al., 2001; Day et al., 2002; Funk et al.,2006; Kim et al., 2006). Interestingly, CRF gene expression inthe dorsal BNST appears to be more dependent onglucocorticoids than that in the ventral BNST (Makinoet al., 1994; Watts and Sanchez-Watts, 1995; Santibanezet al., 2005) and it is possible that the higher (phasic)activity of the HPA axis in RLA-I rats may contribute to theenhanced CRF gene expression in the dorsal BNST. Themechanisms involved in the line-dependent differentialexpression of CRF in the two regions remain to be furtherstudied. Although both regions share projections from theCeA and brainstem nuclei (nucleus tractus solitarius,parabrachial, dorsal raphe, locus coeruleus), they alsoreceive differential projections from other brain areas(Dong et al., 2001) that can be important for themaintenance of a higher CRF gene expression in the dorsal,but not ventral BNSTof RLA-I rats as compared to RHA-I rats.

The BNST is considered to take part of the brain circuitsinvolved in the control of anxiety (e.g. Davis et al., 1993;Gray and McNaughton, 2000; McNaughton and Gray, 2000).The present data suggest that enhanced activity of CRF+neurons in the dorsal BNST may be related to increasedanxiety in RLA-I rats, as this strain shows elevated levels ofcontext and cue-conditioned freezing (i.e. conditioned fear)as well as enhanced fear (cue)-potentiated startle responses(Lopez-Aumatell et al., 2005). It is noteworthy that chronicsocial stress in the visible burrow system has been reportedto increase CRF mRNA levels in the dorsal but not ventralBNST of subordinate animals (Choi et al., 2006), suggestingthat RLA-I rats may have behavioral characteristics similarto those of subordinate animals.

The activity of the HPA axis is under the control ofglucocorticoids through negative feedback mechanismsexerted at different levels: anterior pituitary corticotropes,PVN, hippocampal formation and mPFC (Armario, 2006). Wethus studied MR and GR gene expression in critical brainareas and found no differences between the two strains.Although this may suggest that the main differences in theregulation of the HPA axis in the two strains are likely toinvolve mechanisms different from negative feedback,previously lower levels of GR binding in the anteriorpituitary and of MR binding in the hippocampus have beenreported (Walker et al., 1989). Therefore, it is possible thatfunctional differences in MR and GR may exist that are notdetected by the study of gene expression. In this regard,very recently, similar degree of inhibition of basal levels ofcorticosterone in the two outbred Roman lines has beenreported after dexamethasone administration, but anenhanced corticosterone response of RLA rats to thecombined dexamethasone-CRF test (Steimer et al., 2007),which appears to be more reliable than dexamethasonealone test in depression (Ising et al., 2005). Unfortunately,the precise mechanisms underlying such altered responseare not known and more studies are needed to characterizealtered regulation of the HPA axis with these two lines/strains. The study of the ontogeny of the enhanced CRF geneexpression and HPA responsiveness to stress may be of greatinterest, taking into account the possibility that suchincreased responsiveness may result from superimposed(mild) environmental stressful influences upon a vulnerablegenetic background.

In summary, the present study has characterized inthe same set of animals several critical componentsof the CRF system and the HPA axis in the inbred Romanstrains, including the response of peripheral HPA hormonesto stress and CRF, GR and MR gene expression in keybrain areas. Our results suggest that the increased HPAresponsiveness to stress of RLA-I as compared to RHA-I rats islikely to be related to an enhanced CRF gene expression inthe PVN, though not linked to altered negative glucocorti-coid feedback mechanisms. In addition, the higher CRFexpression in the dorsal BNST may also be important inexplaining other fear-related differences between the twostrains.

Role of the funding source

Sponsors of this study had no role in study design; in thecollection, analysis and interpretation of data; in the writingof the report; and in the decision to submit the paper forpublication.

Conflict of interest

None declared.

Acknowledgments

This work was supported by Grants SAF2005-00358 (MEC)and RD06/0001/0015 (Instituto de Salud Carlos III, Redestematicas de Investigacion Cooperativa en Salud) to A.A.,

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SAF2003-03480 to A.F.-T. and 2005SGR-00885 to A.T. andA.F.-T and EURATools (AFT; European Commission Contractno LSHG-CT-2005-019015).

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