The Flinders Sensitive Line rats, a genetic model of depression, show abnormal serotonin receptor mRNA expression in the brain that is reversed by 17β-estradiol

Molecular Brain Research 74 Ž1999. 158–166 www.elsevier.comrlocaterbres

Research report

The Flinders Sensitive Line rats, a genetic model of depression, show abnormal serotonin receptor mRNA expression in the brain that is reversed by 17b-estradiol a ¨ Marie K. Osterlund , David H. Overstreet b, Yasmin L. Hurd a

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Department of Clinical Neuroscience, Psychiatry Section, Karolinska Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden b Department of Psychiatry, UniÕersity of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7175, USA Accepted 31 August 1999

Abstract The possible link between estrogen and serotonin Ž5-HT. in depression was investigated using a genetic animal model of depression, the Flinders Sensitive Line ŽFSL. rats, in comparison to control Flinders Resistant Line rats. The mRNA levels of the estrogen receptor ŽER. a and b subtypes and the 5-HT1A and 5-HT2A receptors were analyzed in several limbic-related areas of ovariectomized FSL and FRL rats treated with 17b-estradiol Ž0.15 mgrg. or vehicle. The FSL animals were shown to express significantly lower levels of the 5-HT2A receptor transcripts in the perirhinal cortex, piriform cortex, and medial anterodorsal amygdala and higher levels in the CA 2–3 region of the hippocampus. The only significant difference between the rat lines in ER mRNA expression was found in the medial posterodorsal amygdala, where the FSL rats showed lower ERa expression levels. Overall, estradiol treatment increased 5-HT2A and decreased 5-HT1A receptor mRNA levels in several of the examined regions of both lines. Thus, in many areas, estradiol was found to regulate the 5-HT receptor mRNA expression in the opposite direction to the alterations found in the FSL rats. These findings further support the implication of 5-HT receptors, in particular the 5-HT2A subtype, in the etiology of affective disorders. Moreover, the ability of estradiol to regulate the expression of the 5-HT1A and 5-HT2A receptor genes might account for the reported influence of gonadal hormones in mood and depression. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Affective disorder; mRNA; Estrogen; In situ hybridization; Limbic system

1. Introduction Epidemiological studies have shown that depression is the most common mental disorder and the lifetime prevalence rate of major depression is approximately 4% in men and 8% in women Žsee reviews w30,51x.. The reason for this gender difference is not clear, but associations to factors related to expression of distress, hormonal effects, and social situation have been hypothesized w50x. Increased prevalence of depression during the reproductive years and the potential for depressive episodes to occur at times of changes in gonadal hormone levels suggests that these hormones play an important role in affective disorders. Depressive disorders such as premenstrual syndrome, postnatal depression, and postmenopausal depression are associated with low levels of estrogens, and hormone replace) C orresponding author. [email protected]

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ment therapy has been shown to improve and prevent the two latter disorders w18,58x. The estrogenic effects are most likely mediated through the estrogen receptors ŽERs.; today two ER subtypes are known, the ERa and ERb. Both receptor subtypes are expressed mainly in limbic associated structures Že.g., the hypothalamus, amygdala, and hippocampus. w41,57x. To date, no studies on the role of ERa and ERb in depression have been reported. Of the neurotransmitter systems studied in the pathophysiology of affective disorders hitherto, an imbalance in the serotonin Ž5-HT. system has been strongly implicated w4,37,66x. Both postmortem analyses w12,21x and in vivo imaging w5x of brain tissue from subjects diagnosed with major depression have reported alterations in 5-HT receptor densities. Moreover, drugs acting on the 5-HT system are widely used in the treatment of affective disorders. Interestingly, estrogen has been shown to regulate the expression of several components of the 5-HT system, such as the 5-HT1A receptor w42,43x, 5-HT2A receptor

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¨ M.K. Osterlund et al.r Molecular Brain Research 74 (1999) 158–166

w14,64x, and 5-HT transporter w36,52x. High estrogen levels are normally associated with decreased 5-HT1A and increased 5-HT2A receptor binding and mRNA levels in the rat brain w14,42,43,64x. To investigate the possible role of estrogen and 5-HT in depression, experimental studies were currently performed in a genetic animal model of depression, the Flinders Sensitive Line ŽFSL. rats. The FSL rats and their corresponding controls, the Flinders Resistant Line ŽFRL. rats, were established by selective breeding for high and low sensitivity, respectively, to the anticholinesterase agent, diispopropyl fluorophosphate w47x. This rat line was originally developed based on the fact that an overactive cholinergic system had been proposed in depression w22x and affective disorder patients showed increased sensitivity to cholinergic agonists w23,55x. The FSL animals show reduced locomotion, disturbed REM sleep, greater degree of ‘‘anhedonia’’ in response to chronic mild stress, cognitive difficulties, and reduced body weight Žsee reviews w44,45x.. Furthermore, the FSL rats respond to chronic, but not acute, antidepressant treatment with desipramine, imipramine, sertraline, or fluoxetine w44,45x. Thus, the FSL rats meet the criteria of face, construct, and predictive validity for an animal model of depression. In regard to the 5-HT system, decreased 5-HT function has been generally hypothesized in depression based on reduced levels of 5-HT and its metabolite 5-hydroxyindoleacetic acid in the CSF or blood of depressed human subjects w3,54x. Post mortem studies of brain specimens from depressed human subjects show, however, an increase Žin the amygdala. w10x or no change w10,13,40x of 5-HT or its metabolites. In the FSL rats, 5-HT and 5-hydroxyindoleacetic acid were found to be elevated in limbic regions and these alterations have been normalized after chronic antidepressant Ždesipramine. treatment w69x. To further characterize the 5-HT system in the FSL animal model of depression, the mRNA expression of the 5-HT1A and 5-HT2A receptor were assessed in discrete brain areas of the FSL and FRL lines in the present study. In addition, the proposed 5-HT–estrogen interaction in depression was evaluated by studying the brain ERa and ERb gene expression in the different rat lines as well as the effects of 17b-estradiol on the 5-HT1A and 5-HT2A receptor mRNA expression. As limbic structures are hypothesized to play a crucial role in the regulation of mood and affect w29,48x and a limbic-cortical dysregulation has been proposed in depression w35x, the mRNA expression levels were primarily studied in brain regions included in the limbic system. 2. Material and methods 2.1. Animals FSL and FRL rats ŽSprague–Dawley derived. were bred and maintained at the Animal facilities of the Department

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of Pharmacology at the Karolinska Institute, Stockholm, Sweden. Twelve FRL Ž224–252 g. and 12 FSL Ž164–200 g. 2.5-month-old female rats were maintained on a 12 h lightrdark schedule at 198C and had free access to food pellets and water. The experimental procedures were carried out in accordance with the guidelines approved by the Stockholm animal ethical committee. The animals underwent bilateral ovariectomy Žanaesthetized with sodium pentobarbital 60 mgrg. and were allowed to recover for 1 week. A subcutaneous injection of 0.15 mgrg body weight 17b-estradiol dissolved in arrachis oil Ž150 mgrml. or arrachis oil vehicle was administered. The animals were decapitated 24 h after the injection and the brains were quickly removed, frozen on dry ice, and stored at y708C. The brains were sectioned Žcoronal. at 20 mm using a cryostat ŽJung-Frigocut 2800 E cryostat, Leica., thawmounted onto glass slides Žcoated with poly-L-lysine., dried at 378C and then stored at y208C. Plasma was also taken at the time of decapitation and estradiol levels measured by standard routine procedures Žusing DELFIA. at the Dept. of Clinical Chemistry at the Karolinska Hospital. The assay detection limit was 50 pM. 2.2. Probe preparations and in situ hybridization Chemicals used in the hybridization procedure were purchased mainly from Sigma ŽMO, USA. and the enzymes from Life Technologies ŽRenfrewshire, Scotland, UK.. The plasmids used for generating rat ERa and ERb specific riboprobes have previously been described w41x. The ERa plasmid contains a 850 bp cDNA insert corresponding to the E and F domains of the receptor and part of the 3X-UTR. The ERb plasmid contains a 355 bp insert encoding part of the 5X-UTR and ArB domain of rat ERb cDNA. The 5-HT1A plasmid Žkindly provided by Dr. P.R. Albert, University of Ottawa. contains a genomic fragment of the rat 5-HT1A receptor gene subcloned into the HincII site of the pGEMBlue vector. The 5-HT2A receptor riboprobe was generated from a pGEM-T Easy plasmid ŽPromega. containing a 300 bp insert of the mouse 5-HT2A receptor cDNA Žnt 1–300 Genebank accession number S-49542; gift of Dr. G.G.J.M. Kuiper, Erasmus University Medical School, Rotterdam.. Sense and antisense riboprobes were transcribed from the linearized plasmids Ž0.5 mg. using the appropriate RNA polymerase ŽSP6, T3, or T7. in the presence of RNase inhibitor, 10 mM dithiothreitol, 0.5 mM each of ATP, GTP, CTP, and 150 mCi a-w35 SxUTP ŽDupont NEN, Boston USA. in a 1 = transcription buffer for 60 min at 378C followed by 10 min of DNase treatment at 378C. The labeled riboprobes were then separated from unincorporated nucleotides using spin columns S-200 HR ŽAmersham Pharmacia Biotech, Uppsala Sweden.. Sections were brought to room temperature and fixed in 4% paraformaldehyde in 1 = phosphate-buffered saline

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ŽPBS; pH 7.4. for 5 min, rinsed two times in 1 = PBS, and once in a TEA buffer Ž0.1 M triethanolamine, 0.9% NaCl, pH 8.0.. This was followed by a 10 min treatment with 0.25% acetic anhydride in TEA buffer and a rinse in 2 = standard saline citrate buffer ŽSSC.. Sections were then processed through graded series of ethanol Ž70%, 80%, 95%, 100%; 1 min. and chloroform Ž5 min. followed by 100% Ž1 min., 95% Ž1 min. ethanol washes and subsequently air dried. The riboprobe hybridization was carried out as follows: heat denatured 35 S-labeled riboprobes were added to the hybridization cocktail Ž50% formamide, 0.5 mg sheared salmon sperm DNA, 0.25 mg yeast tRNA, 1 = Denhardt solution, 4 = SSC, and 200 mM dithiotreitol. to a final concentration of 2000 c.p.m. per mm2 , and 0.1 ml per mm2 of cocktail was applied to each section. The sections were coverslipped to prevent evaporation and the hybridization was carried out in a humidified chamber overnight at 558C. After hybridization, the sections were rinsed in 2 = SSC followed by RNAse A treatment Ž40 mgrml. for 30 min at 378C. The sections were then washed in graded series of SSC Ž2 = 10 min, 1 = 10 min, 0.5 = 10 min, 0.5 = r50% formamide 1 h 488C, 0.1 = 1 h 538C. containing 1 mM dithiotreitol; all at room temperature except for the formamide and 0.1 = SSC washes. Subsequently, the sections were dehydrated with ethanol containing 300 mM ammonium acetate. The slides were then air dried and exposed to Amersham b-max Hyperfilm with 14 C standards ŽAmerican Radiolabeled Chemicals, MO, USA. for 2–3 weeks.

Results were considered significant for p F 0.05 and a trend was considered for p F 0.10.

2.3. Image analyses Autoradiograms were scanned at a resolution of 300 dpi with a ScanMaker II ŽMicrotek Electronics, Dusseldorf, ¨ Germany.. Light transmittance values were measured from the digitalized images in the Macintosh-based image analysis software system ŽIMAGE; Wayne Rasband, NIMH, Bethesda, MD.. The light transmittance values were converted to dpmrmg tissue using the co-exposed standards. Coronal brain sections were examined at approximate levels 1.7 to y4.16 mm relative to Bregma in conjunction with Paxinos and Watson stereotaxic rat brain atlas w49x. Limbic-related brain areas of interest included the hippocampus, hypothalamus, amygdala, accumbens Žshell., cingulate cortex Žareas 1 and 2., perirhinal cortex, and piriform cortex. In addition, a non-limbic cortical area Žmotor cortex Žareas 1 and 2.. was also examined. Correlation between body weight and dpmrmg values was tested for each rat line ŽFSL and FRL. using the Pearson correlation test. Statistical evaluations of the 17b-estradiol treatment and differences between the rat lines in each brain area of interest were assessed by analyses of variance Žtwo-way ANOVA; rat line and treatment with rat line by treatment interaction.. All statistical analyses were carried out using the JMP Ž3.1 v. statistical software package.

Fig. 1. Hybridization signals obtained using the antisense riboprobes complementary to the ERa ŽA., ERb ŽB., 5-HT1A receptor ŽC., and 5-HT2A receptor ŽD. mRNA in a respective vehicle treated FRL Žleft panel. and FSL Žright panel. rat. The brain sections shown are approximately y3.1 mm from Bregma. Scale bar s 2 mm. Abbreviations: Arc, arcuate nucleus; VMH, ventromedial hypothalamic nucleus; PLCo, posterolateral cortical amygdala nucleus; MePD, medial posterodorsal amygdala nucleus; Mo, motor cortex; DG, dentate gyrus; PRh, perirhinal cortex; CA 2–3, fields of hippocampus; Pir, piriform cortex.

¨ M.K. Osterlund et al.r Molecular Brain Research 74 (1999) 158–166

3. Results In situ hybridization using the different antisense probes Žcomplementary to the ERa , ERb, 5-HT1A receptor, and 5-HT2A receptor mRNA. resulted in distinct distribution patterns consistent with previous reports w9,41,53x ŽFig. 1.. No signals above background were observed with the corresponding sense probes. In agreement with previous observations, the FRL and FSL rats differed significantly in weight w44x, but no correlation between body weight and dpmrmg was found in any of the brain areas examined. 17b-estradiol was still present in the blood Ž192 " 50 pM. 24 h after the estradiol injection, whereas the vehicle-administered animals showed no detectable 17b-estradiol levels. 3.1. ER a mRNA expression ERa mRNA was highly expressed in the ventromedial hypothalamus ŽFig. 1A., arcuate nucleus ŽFig. 1A., medial amygdala ŽFig. 1A., amygdala hippocampal area, and posterolateral cortical amygdala ŽFig. 1A.. A significant difference for the ERa mRNA expression was found between the rat lines in the medial posterodorsal amygdala nucleus, with lower transcript levels Ž7.7%. in the FSL as compared to the FRL rats Ž F1,21 s 4.557; p - 0.05.. In addition, a trend for lower levels in the ventromedial hypothalamus Ž F1,21 s 4.000; p - 0.07. of the FSL rats was also detected ŽFig. 2A.. Compared to vehicle-treated

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control animals, 17b-estradiol treatment significantly decreased ERa mRNA expression in all brain areas examined, except for the amygdala hippocampal area ŽFig. 2B.. To validate the effectiveness of the estradiol treatment, the ventromedial hypothalamus and the arcuate nucleus were measured as control areas since a down-regulation of ERa mRNA expression after 17b-estradiol treatment is well documented for these areas w27,41,59x. Consistent with the previous studies, the ERa mRNA was reduced by 17bestradiol. The reduction ranged between 7.0% and 11.9% in following areas: the arcuate nucleus Ž F1,17 s 10.442; p - 0.006., ventromedial hypothalamus Ž F1,20 s 14.453; p - 0.002., medial anterodorsal amygdala Ž F1,21 s 4.481; p - 0.05., medial posterodorsal amygdala Ž F1,21 s 10.341; p - 0.005., and posterolateral cortical amygdala Ž F1,21 s 8.815; p - 0.008.. No interaction between the rat line and 17b-estradiol treatment was found. 3.2. ER b mRNA expression The statistical analyses showed no significant effect of the rat line, 17b-estradiol treatment, or rat line by treatment interaction in the ERb mRNA levels in the brain regions analyzed: the paraventricular nucleus, supra optic nucleus, medial anterodorsal amygdala, medial posterodorsal amygdala, and the hippocampus Ždata not shown.. 3.3. Serotonin 5-HT1 A mRNA expression The 5-HT1A receptor mRNA showed moderate to high expression in areas associated with the regulation of emo-

Fig. 2. ERa mRNA expression levels in different brain areas of the FSL ŽA. and the 17b-estradiol Ž0.15 mgrg. treated ŽB. rats, compared to FRL and vehicle Žoil. treated control animals, respectively. Bars represent the mean" S.E.M. Ž n s 11–12. values of the hybridization signal Ždpmrmg., given as a percentage of the control FRL rats Žset to 100%; Arc s 690.9 " 13.9; VMH s 622.8 " 7.9; MeADs 124.7 " 3.3; MePDs 503.6 " 15.2, AHiA s 605.5 " 34.3; PLCos 492.3 " 12.2 dpmrmg. ŽA. and of the control oil vehicle treated rats Žset to 100%; Arc s 597.2 " 8.4; VMH s 630.8 " 5.8; MeADs 129.52 " 3.2; MePDs 514.2 " 16.1, AHiA s 617.2 " 35.4; PLCo s 525.9 " 11.6 dpmrmg. ŽB.. U p - 0.05; UU p - 0.01. Abbreviations: Arc, arcuate nucleus; VMH, ventromedial hypothalamic nucleus; MeAD, medial anterodorsal amygdala nucleus; MePD, medial posterodorsal amygdala nucleus; AHiA, amygdala hippocampal area; PLCo, posterolateral cortical amygdala nucleus.

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Fig. 3. 5-HT1A receptor mRNA expression levels in different brain areas of the FSL ŽA. and the 17b-estradiol Ž0.15 mgrg. treated ŽB. rats, compared to FRL and vehicle Žoil. treated control animals, respectively. Bars represent the mean" S.E.M. Ž n s 11–12. values of the hybridization signal Ždpmrmg., given as a percentage of the control FRL rats Žset to 100%; Cg s 278.0 " 9.1; Mo s 276.8 " 8.5; PRh s 188.1 " 4.2; Pir s 403.8 " 13.4, PMCos 429.7 " 12.7; MeADs 192.3 " 6.5; CA 2–3 s 647.5 " 5.9; DG s 760.9 " 8.2 dpmrmg. ŽA. and of the control oil vehicle treated rats Žset to 100%; Cg s 295.5 " 5.7; Mo s 290.85 " 7.7; PRh s 199.7 " 5.6; Pir s 430.8 " 12.4, PMCos 441.2 " 12.3; MeADs 212.0 " 3.8; CA 2–3 s 645.8.3 " 4.0; DG s 761.2 " 9.2 dpmrmg. ŽB.. U p - 0.05; UU p - 0.01; UUU p - 0.001; UUUU p - 0.0001. Abbreviations: Cg, cingulate cortex; Mo, Motor cortex; PRh, perirhinal cortex; Pir, piriform cortex; PMCo, posteromedial cortical amygdala; MeAD, medial anterodorsal amygdala nucleus CA 2–3, fields of hippocampus; DG, dentate gyrus.

tion, memory, and cognition, such as the amygdala, hippocampus, and cerebral cortex ŽFig. 1C.. There were no significant differences in 5-HT1A mRNA expression between the two rat lines in the brain areas examined, although a trend for higher transcript levels in the FSL rats

was observed in the perirhinal cortex Ž F1,21 s 3.098; p 0.09. and the medial anterodorsal amygdala Ž F1,21 s 3.211; p s 0.09. ŽFig. 3A.. The 17b-estradiol treatment reduced 5-HT1A mRNA levels in several areas ŽFig. 3B.. Compared to vehicle-treated control animals, the expression

Fig. 4. 5-HT2A receptor mRNA expression levels in different brain areas of the FSL ŽA. and the 17b-estradiol Ž0.15 mgrg. treated ŽB. rats, compared to FRL and vehicle Žoil. treated control animals, respectively. Bars represent the mean" S.E.M. Ž n s 11–12. values of the hybridization signal Ždpmrmg., given as a percentage of the control FRL rats Žset to 100%; AcbShs 106.5 " 3.4; Cg s 121.1 " 6.0; Mo s 141.2 " 7.7; PRh s 142.3 " 6.6; Pir s 133.7 " 2.5, MeADs 66.43 " 2.4; CA 2–3 s 103.3 " 1.2 dpmrmg. ŽA. and of the control oil vehicle treated rats Žset to 100%; AcbShs 101.8 " 3.8; Cg s 113.8 " 6.9; Mo s 128.0 " 7.2; PRh s 118.3 " 7.4; Pir s 121.52 " 3.4; MeADs 58.88 " 2.9; CA 2–3 s 112.7 " 3.3 dpmrmg. ŽB.. U p - 0.05; UU p - 0.01; UUU p - 0.001; UUUU p - 0.0001. Abbreviations: AcbSh, nucleus accumbens shell; Cg, cingulate cortex; Mo, motor cortex; PRh, perirhinal cortex; Pir, piriform cortex; MeAD, medial anterodorsal amygdala nucleus; CA 2–3, fields of hippocampus.

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levels were significantly decreased Ž10.5%–14.5%. in the cingulate cortex Ž F1,21 s 8.359; p - 0.01., the motor cortex Ž F1,21 s 19.240; p - 0.0003., the piriform cortex Ž F1,21 s 7.994; p ) 0.02., and the medial anterodorsal amygdala nucleus Ž F1,21 s 34.376; p - 0.0001. in the 17b-estradiol treated animals. There were no significant treatment differences in the other brain regions examined: perirhinal cortex, posteromedial cortical amygdala, CA 2–3 region of the hippocampus, or the dentate gyrus. No significant interactions were observed between the rat line and 17bestradiol treatment. 3.4. Serotonin 5-HT2 A mRNA expression Moderate to low expression levels of the 5-HT2A receptor mRNA were evident in the hippocampus ŽFig. 2D., cerebral cortex ŽFig. 2D., and striatum. Differences between the rat lines in 5-HT2A mRNA expression were found in a number of the brain areas examined ŽFig. 4A.. Significantly lower 5-HT2A mRNA expression levels Ž9.9%–20.8%. were evident in the perirhinal cortex Ž F1,21 s 13.813; p - 0.002., piriform cortex Ž F1,21 s 15.361; p - 0.0009., and medial anterodorsal amygdala nucleus Ž F1,21 s 15.586; p - 0.0009. in the FSL rats. In contrast, a highly significant increase Ž23.4%. of the 5-HT2A mRNA levels Ž F1,21 s 173.468; p - 0.0001. was evident in the CA 2–3 region of the hippocampus in the FSL as compared to the FRL rats. No significant rat line differences were observed in the nucleus accumbens shell, cingulate cortex, or motor cortex. Following estradiol treatment, a significant increase in the 5-HT2A mRNA expression Ž4.0%–17.3%. was detected in the nucleus accumbens shell Ž F1,21 s 5.308; p - 0.04., perirhinal cortex Ž F1,21 s 6.473; p - 0.02., piriform cortex Ž F1,21 s 13.931; p 0.002., and CA 2–3 Ž F1,21 s 9.714; p - 0.006. ŽFig. 4B.. No significant treatment effects were observed in the cingulate cortex, motor cortex, or medial anterodorsal amygdala nucleus. A trend for an interaction between the rat line and 17b-estradiol treatment was evident in the piriform cortex, hippocampus, and nucleus accumbens, due to a greater estradiol effect in the FSL rats. 4. Discussion In the current study, abnormalities in the 5-HT2A receptor and the ERa mRNA expression were detected in the FSL rats, a genetic animal model of depression. Furthermore, in several brain areas, 17b-estradiol treatment was shown to reverse the alterations found in the 5-HT2A mRNA expression supporting an estrogen–5-HT interaction in affective disorders. The 5-HT2A receptor has been frequently studied in post mortem human brains of subjects diagnosed with major depression and of suicide victims. The main results indicate increase w2,21,61x, decrease w11,19x, or no change w11,32,62x in 5-HT2 receptor binding in the frontal cortex and hippocampus as compared to

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control subjects. These conflicting post mortem findings may be due to differences in diagnosis and drug treatments in the subjects studied as well as in the receptor binding methods used. In the drug-naive FSL rats, the mRNA expression levels of the 5-HT2 receptor mRNA levels were reduced in several of the limbic cortical regions examined. It could be speculated that alterations in mRNA expression levels are not correlated to changes at the level of the receptor protein. However, though the time course between mRNA and protein alterations may differ, the direction of change of these alterations is usually similar. Consistent with a reduction of the 5-HT2 receptor mRNA expression in the FSL rats, in vivo analyses of drug-free depressed patients using positron emission tomography have in fact shown reduced levels of 5-HT2 receptors in limbic related cortical areas Žorbitofrontal and anterior insula. w5x. In addition to the 5-HT2A receptor, there is convincing evidence for the involvement of the 5-HT1A receptor in depression and anxiety w15,31x. Although no significant differences Žonly trends for increase. in 5-HT1A receptor mRNA expression were evident in the FSL as compared to the FRL rats, the role of the 5-HT1A receptor in affective disorders cannot be discounted. It has been previously demonstrated that the FSL rats have a supersensitivity to 5-HT1A receptor agonists w46x. Moreover, several groups have examined 5-HT1A receptor densities in post mortem brain tissue of suicide victims and subjects diagnosed with major depression. In the majority of these studies, the 5-HT1A receptor levels Žin hippocampus and cerebral cortex. were reported to be increased w1,24,34x or unaltered w12,62x as compared to control subjects. Increased 5-HT1A autoreceptors in the midbrain dorsal raphe nucleus have also been revealed in suicide victims diagnosed with major depression w63x. Enhanced 5-HT1A receptor activity further support the hypothesized 5-HT-depletion in depression since primarily presynaptic, but also postsynaptic, 5-HT1A receptors have been shown to inhibit the firing of 5-HT neurons w15x. This is the first study to our knowledge to use an animal model of depression to investigate the possible role of estrogen in affective disorders. Interestingly, the 5-HT2A receptor mRNA expression abnormalities found currently in the FSL animals were reversed in several of the affected brain areas by the 17b-estradiol treatment. In the estradiol treated animals, the 5-HT2A receptor transcripts were significantly increased in the accumbens shell, perirhinal cortex, piriform cortex, and CA 2–3 region. Previous studies have shown both the 5-HT2A receptor mRNA w14x and protein w14,64x levels to be up-regulated in the nucleus accumbens and cerebral cortex by estradiol treatment, or down-regulated after ovariectomy. In the present study, a marked effect of the estradiol treatment on the 5-HT1A receptor mRNA levels in the cingulate cortex, motor cortex, piriform cortex, and the medial anterodorsal amygdala. These treatment effects are consistent with our previous findings on altered 5-HT1A receptor mRNA w43x and pro-

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tein w42x levels following estradiol administration. Even though estradiol regulates the 5-HT1A and 5-HT2A receptor expression in opposite directions, the 5-HT cellular and behavioral response may not be divergent. In fact, activation of the two receptor families Žthe 5-HT1 and 5-HT2 . often produce opposite actions w56,65x and the two receptor subtypes are coupled to different second messenger systems w31,65x. In considering the link between affective disorders and low serum–estrogen levels, one would expect estrogen to exhibit antidepressant properties and thereby mimic the 5-HTergic receptor changes associated with antidepressant treatment. Consistent with the present findings, electroconvulsive stimulation Žwhich is an animal model for electroconvulsive antidepressant therapy. increased 5-HT2 receptor levels in the cerebral cortex w7,25x. In addition, fluoxetine treatment has been shown to up-regulate the 5-HT2 receptor binding in the frontal cortex, both in depressive patients Žexamined with positron emission tomography. w33x and in rats w20x. However, other studies have shown reduced 5-HT2 receptor levels in the rat cerebral cortex after treatment with antidepressant drugs w17,67x. As in the case for the 5-HT2 receptor, antidepressant drug treatment has been shown to regulate 5-HT1A receptor densities in different directions w6,26,39x or not at all w67x. In part, these differences appear to depend on which antidepressant drug and which rat strain that have been studied w6,26x. Although there are not as yet any consistent alteration of the 5-HT-receptor densities following antidepressant treatments, most of the therapies do have a common feature of affecting limbic-related 5-HT neuronal populations. Despite a role of estrogen in depression, differences between the FRL and FsL rats in the mRNA expression of the ERs were minimal. A significant difference in the ERa mRNA expression was only detected in the medial posterodorsal amygdala. This region expressed lower levels of ERa mRNA in the FSL as compared to the FRL rats. The posterodorsal amygdala is included in the limbic–hypothalamic circuit that is known to regulate reproductive behavior w38,60x. In contrast with the lower ERa mRNA levels found in the present study, the female FSL rats have been shown to exhibit enhanced sexual behavior in response to estrogen combined with progesterone, as compared to FRL and Long Evans control rats, suggesting a supersensitivity for gonadal hormones w16x. The present results indicate that this behavioral hormone sensitivity is, however, not due to increased ER gene expression. In addition to similar basal ER mRNA expression levels between the rat lines, there was a similar responsivity of the ERa mRNA expression to the 17b-estradiol treatment. In agreement with the literature w28,41,59x, the ERa receptor mRNA was significantly decreased by the estradiol treatment in several hypothalamic and amygdaloid areas. Thus, the present findings in the FSL genetic animal model of depression would suggest that the estrogenic influence in depression is not defined at the level of expression or

regulation of the ER mRNA subtypes, but more in the ability of estradiol to regulate components of the 5-HT system. Although, behaviorally, the FSL rats do meet the criteria of face, construct, and predictive validity of an animal model of depression, further studies of this relatively new animal model are necessary. Thus far, impairment of the 5-HT ŽRef. w69x; present results., neuropeptide Y w8x, catecholamine w68x, and cholinergic w44x systems have been detected in the FSL rats, neurochemical systems shown to be abnormal in human depressed subjects. The fact that several of these neurochemical abnormalities in the FSL rats have been normalized following chronic antidepressant treatments w8,68,69x provides strong evidence for the predictive validity of this animal model of depression. How the neurobiological alterations observed in this animal model specifically relate to the psychiatric disorder is unclear since human studies have not as yet provided detailed information as to the discrete neuronal populations that are impaired in depression. More detailed post mortem and in vivo imaging studies of drug naive human subjects will help to assess if the neurobiological changes observed in the FSL animals are truly reflective of depression. Taken altogether, significant alterations in the 5-HT2A receptor mRNA levels and trends for increased 5-HT1A receptor transcripts in several limbic-related areas point to impaired 5-HT function in the ‘‘depressed’’ FSL rats. The current results also strongly support an estrogen–5-HT interaction considering that the estradiol treatment effects in several brain areas counteracted the alterations that were found for the 5-HT receptor gene expression in the FSL rats. These findings suggest that the 17b-estradiol regulation of the 5-HT receptors, at least in part, is a possible underlying mechanism in the link between estrogen and depression. Further studies are required to assess if estradiol treatment is also able to affect any of the impaired behavioral functions associated with depression in the FSL rats. Acknowledgements We thank Mrs. Siv Eriksson for technical assistance. This work was supported by grants from Kapten Arthur Erikssons Stiftelse and the Karolinska Institutes Stiftelse. References w1x V. Arango, M.D. Underwood, A.V. Gubbi, J.J. Mann, Localized alterations in pre- and postsynaptic serotonin binding sites in the ventrolateral prefrontal cortex of suicide victims, Brain Res. 688 Ž1995. 121–133. w2x R.C. Arora, H.Y. Meltzer, Serotonergic measures in the brains of suicide victims: 5-HT2 binding sites in the frontal cortex of suicide victims and control subjects, Am. J. Psychiatry 146 Ž1989. 730–736. ˚ w3x M. Asberg, L. Bertilsson, B. Martensson, G.P. Scalia, P. Thoren, L. ˚ Traskman-Bendz, CSF monoamine metabolites in melancholia, Acta ¨ Psychiatr. Scand. 69 Ž1984. 201–219.

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