Effect of neonatal handling on serotonin 1A sub-type receptors in the rat hippocampus

Neuroscience 140 (2006) 1–11

EFFECT OF NEONATAL HANDLING ON SEROTONIN 1A SUB-TYPE RECEPTORS IN THE RAT HIPPOCAMPUS A. STAMATAKIS, A. MANTELAS, A. PAPAIOANNOU, S. PONDIKI, M. FAMELI AND F. STYLIANOPOULOU*

effects of handling as to the vulnerability toward depression-like behavior in response to chronic stressful stimuli. © 2006 Published by Elsevier Ltd on behalf of IBRO.

Department of Basic Sciences, Faculty of Nursing, School of Health Sciences, University of Athens, 123 Papadiamantopoulou str., 11527 Athens, Greece

Key words: 5-HT1A receptors, early handling, sex differences, in situ hybridization, immunohistochemistry, in vitro binding.

Abstract—Serotonin 1A sub-type receptors play an important role in the etiopathogenesis of depression, which is known to occur more often in females than males. Early experiences can be a predisposing factor for depression; however, the underlying cellular processes remain unknown. In an effort to address such issues, we employed neonatal handling, an experimental model of early experience, which has been previously shown to render females more vulnerable to display enhanced depression-like behavior in response to chronic stress, while it increases the ability of males to cope. In rat pre-pubertal (30 days of age) and adult (90 days) hippocampus, of both males and females, the effect of neonatal handling on serotonin 1A sub-type receptor mRNA and protein levels was determined by in situ hybridization and immunohistochemistry, respectively, while the number of binding sites was determined by in vitro autoradiography using [3H]8-hydroxy2(di-n-propylamino)tetralin as the ligand. Our results revealed a significant sex difference in serotonin 1A sub-type receptor mRNA, protein and binding sites, with females having higher levels than males. Handling resulted in statistically significant decreased numbers of cells positive for serotonin 1A sub-type receptor mRNA or protein, as well as [3H]8-hydroxy-2(di-n-propylamino)tetralin binding sites in the area 4 of Ammon’s horn and dentate gyrus of both pre-pubertal males and females. In adult animals the number of serotonin 1A sub-type receptor mRNA positive cells was increased as a result of handling in the area 1 of Ammon’s horn, area 4 of Ammon’s horn and dentate gyrus of males, while it was decreased only in the area 4 of Ammon’s horn of females. Furthermore, the number of serotonin sub-type 1A receptor immunopositive cells, as well as [3H]8-hydroxy-2(di-n-propylamino)tetralin binding sites was increased in the area 1 of Ammon’s horn, area 4 of Ammon’s horn and dentate gyrus of handled males, whereas it was decreased in these same brain areas in the handled females. We can thus infer that neonatal handling results in alterations in postsynaptic serotonergic neurotransmission, which may contribute to the sex dimorphic

Depression is a common psychiatric disease, with a prevalence ranging from 5% to 20% (Hamet and Tremblay, 2005). It is associated with decreased quality of life for patients and their families, and a substantial financial burden on society, both because of workdays lost and cost of treatment. By the year 2020 depression is predicted to be the second greatest disease-burden worldwide (Murray and Lopez, 1996). Epidemiological studies across countries and ethnic groups have shown that depression occurs twice as often in women than men (Noble, 2005). Experimental studies have also shown that females are more vulnerable than males in animal models of depression (Kennett et al., 1986; Chaouloff, 2000). It should be noted that dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis is a characteristic feature of depression. In melancholic depression there is hyperactivity of the HPA axis, while hypoactivity is observed in atypical depression (Gold and Chrousos, 2002; Antonijevic, 2006). Notably, female predominance among patients with depression seems to be restricted to the atypical subtype (Antonijevic, 2006). Previous results from our laboratory have shown that neonatal handling, although in general beneficial, renders females, when adult, more vulnerable to express depressive behavior in paradigms of chronic stress, while it increases the ability of males to actively cope (Papaioannou et al., 2002b). Neonatal handling, a form of brief, repeated maternal separation during the first three weeks of life has been shown to alter the programming of HPA axis function in such a way that the ability of the organism—particularly the male—to respond, cope and adapt to stressful stimuli is increased in adulthood (Levine, 1957; Ader and Grota, 1969; Hess et al., 1969; Meaney et al., 1991). As a consequence, adult handled rats show lower ACTH and corticosterone responses to stress, less fear in novel environments, more exploratory behavior, and lower emotionality (Vallée et al., 1997; Fernandez-Teruel et al., 2002). Since, handled females have lower stress-induced plasma corticosterone levels that the non-handled (Levine, 1957; Ader and Grota, 1969; Hess et al., 1969; Meaney et al., 1991; Vallée et al., 1997), it appears that the depressive-like behavior of the handled females resembles more the atypical form of depression.

*Corresponding author. Tel: ⫹30-210-7461453; fax: ⫹30-210-7461489. E-mail address: [email protected] (F. Stylianopoulou). Abbreviations: ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; Bmax, maximum binding sites; CA1– 4, areas 1– 4 of Ammon’s horn; DG, dentate gyrus; DIG, digoxigenin; GR, glucocorticoid receptor; HPA, hypothalamic–pituitary–adrenal; Kd, equilibrium dissociation constant; MR, mineralocorticoid receptor; NGS, normal goat serum; PBS, phosphate-buffered saline; PND, postnatal day; RT, room temperature; S.D., standard deviation; SSC, standard saline citrate buffer; TBS, Tris-buffered saline; [3H]-8-OH-DPAT, [3H]8-hydroxy-2(di-n-propylamino)tetralin; 5-HT, serotonin; 5-HT1A, serotonin 1A sub-type receptor; 5-HT2, serotonin 2 sub-type receptor. 0306-4522/06$30.00⫹0.00 © 2006 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2006.01.035

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It has been proposed that the effects of neonatal handling are mediated by alterations in maternal behavior, specifically increased licking and grooming of the pups (Levine, 1994). It should also be noted that, even under normal conditions, mothers spend more time caring for their male than their female offspring (Moore and Morelli, 1979). Thus differences in maternal behavior expressed by the dams toward their male and female offspring could account for the known sex dimorphic effects of neonatal handling (Smythe et al., 1994a; Papaioannou et al., 2002a,b; Park et al., 2003) including the consequences of this early experience on the vulnerability of handled female animals to express, as adults, “depression” when facing chronic stressful stimuli. There is ample evidence supporting a significant functional interaction between the hippocampus–HPA axis and the central serotonergic system in the control of the stress response and mood states such as anxiety and depression. More specifically, the responses of the area 1 of Ammon’s horn (CA1– 4, areas 1– 4 of Ammon’s horn) pyramidal cells to serotonin (5-HT) depend on the levels of circulating corticosteroids and thus the degree of occupation of the two types of hippocampal corticosteroid receptors, glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) (Joels and De Kloet, 1991; Hesen and Joels, 1996). On the other hand expression of GRs in the hippocampus involves the activation of 5-HT 7 sub-type receptors (5-HT7), through which the effects of maternal care are imprinted on the developing hippocampus (Weaver et al., 2001), while the neonatal handling-induced increase in hippocampal GRs is mediated through serotonin 2 sub-type receptors (5-HT2) (Mitchell et al., 1990; Smythe et al., 1994b). Type 1A 5-HT receptors appear to be particularly involved in the organism’s response to stress. High levels of these receptors are found throughout the hippocampus, where they are postsynaptic (Blier and Ward, 2003), and their expression is influenced by glucocorticoids (Chalmers et al., 1993). Furthermore, it is well documented, using several experimental paradigms that this receptor is downregulated following chronic stress (McKittrick et al., 1995; Lopez et al., 1997, 1999; Froger et al., 2004; Griffin et al., 2005). Notably stress can act as an eliciting factor leading to the precipitation of depressive episodes in biologically predisposed individuals. In addition, most animal models of depression involve the response to chronic stress paradigms. This kind of evidence pointed to a role of serotonin 1A sub-type receptors (5-HT1A) in depression. Indeed it was shown that depressed patients have blunted responses to the administration of 5-HT1A receptor agonists in vivo (Cowan, 2000). More importantly 5-HT1A binding and mRNA was reduced in the pars opercularis and temporal pole of depressed patients (Bowen et al., 1989) and in the hippocampus of suicide victims (Cheetham et al., 1990; Lopez et al., 1998) as assessed in postmortem material. Furthermore, in vivo imaging, using positron emission tomography (PET), demonstrated reduced 5-HT1A binding in the hippocampus of depressed patients (Drevets et al., 1999, 2000). It should be noted that sex differences in

brain 5-HT1A receptor levels have been documented (Mendelson and McEwen, 1991; Zhang et al., 1999; Parsey et al., 2002). In an effort to address questions regarding the mechanisms through which neonatal handling affects HPA axis reactivity and through it possibly the vulnerability toward depression, we have determined the effect of neonatal handling on 5-HT1A receptor levels in the rat hippocampus, a major anatomic site of the effects of early handling (Meaney et al., 1989). 5-HT1A receptor levels have been determined not only in the adult (90 day old), but also in the pre-pubertal (30 day) rat brain, since puberty is a developmental phase characterized by intense hormonal and behavioral changes, which could underlie the reported increased occurrence of depressive episodes among adolescents (Birmaher et al., 1996; Angold et al., 1999; Young and Altemus, 2004). Furthermore, the sex difference in the prevalence of depression first appears in adolescence (Nolen-Hoeksema and Girgus, 1994). Hippocampal 5-HT1A receptors were determined in both males and females, given the well-known sex difference in HPA axis function and in the prevalence of depression.

EXPERIMENTAL PROCEDURES Animals Wistar rats of both genders reared in our laboratory were kept under standard conditions (24 °C; 12-h light/dark cycle, lights on at 8:00 am, three animals of the same sex per cage) and received food and water ad libitum. Virgin females were exposed to stud males and pregnancy was determined by the presence of sperm in the vaginal smear (day 0 of pregnancy). Prior to birth litters were randomly assigned to either the handled or non-handled category. The average litter size was 8⫾1 pups (mean⫾standard error of the mean, range: 5–13). Litters were not culled, since it has been shown that litter size within this range (5–18) does not affect maternal behavior (Champagne et al., 2003). The gender ratio did not differ among the litters employed in the different animal groups. The day of birth was defined as postnatal day 0 (PND0). Two cohorts of animals were used in this study: Cohort A was used for the determination of 5-HT1A receptor levels on PND30 (12 male non-handled, 12 male handled, 12 female non-handled, 12 female handled; half of which were used for in situ hybridization and the other half for immunohistochemistry and in vitro autoradiographic binding); Cohort B was used for the determination of 5-HT1A receptor levels on PND90 (12 male non-handled, 12 male handled, 12 female non-handled, 12 female handled; half of which were used for in situ hybridization and the other half for immunohistochemistry and in vitro autoradiographic binding). All animal experimentations were carried out in agreement with ethical recommendation of the European Communities Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize the number of animals used and their suffering.

Neonatal handling The “neonatal handling” protocol employed was as originally described by Levine (1957), which involves removal of the pups from the nest for 15 min daily, during the neonatal period and placing them in a separate container, taking care to control for the pups’ body temperature. In the present experiments “handling” was performed from PND1 until weaning (PND22). Specifically, every day between 9:00 –10:00 a.m. mothers of the pups to be subjected to handling were removed from their

A. Stamatakis et al. / Neuroscience 140 (2006) 1–11 home cages and temporarily placed separately into cages [the same cage for each mother every day for the duration (22 days) of handling]. Their pups were then removed and placed into plastic containers, lined with paper towels. After 15 min the pups, and then their mothers, were returned to their home cages. “Nonhandled” pups were left undisturbed with their mothers in their home cage until weaning.

Tissue preparation Animals used for immunohistochemistry and in vitro binding were deeply anesthetized, decapitated and brains were isolated and frozen in ⫺40 °C isopentane. Ten micrometer sections were cut on a cryostat (Leica CM1900, Nussloch, Germany) at ⫺17 °C, collected on silane-coated slides and stored at ⫺80 °C until further processing. Animals used for in situ hybridization were deeply anesthetized, transcardially perfused with 200 ml of ice-cold 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4), post-fixed overnight in the perfusion medium at 4 °C, further processed and embedded in paraffin. Serial sagittal sections (6 ␮m) were cut and mounted on silane-coated slides.

5-HT1A mRNA in situ hybridization Riboprobe preparation. We used a 383 bp cDNA fragment of the 5-HT1A receptor coding for the third cytoplasmic loop of the rat 5-HT1A receptor (Karten et al., 1999), subcloned into a pSportII plasmid, which was kindly provided by Dr. PapadopoulouDaifoti Z. (Drossopoulou et al., 2004). The SalI-linearized plasmid was used for the synthesis of the digoxigenin (DIG)-UTP-labeled antisense 5-HT1A riboprobe by T7-mediated in vitro transcription. Sense control 5-HT1A probe was concurrently generated by T3mediated in vitro transcription in the presence of DIG-UTP using as template the NotI-linearized plasmid and did not show detectable signal in the hippocampus.

Riboprobe hybridization In situ hybridization histochemistry was carried out according to the protocol described by Breitschopf and Suchanek (1996). Slide-mounted 6 ␮m saggital sections were deparaffinized, rehydrated and washed (2⫻2 min) with Tris-buffered saline (TBS, pH 7.4). Brain sections were then treated with 0.2 N HCl (20 min), washed again (2⫻5 min) with TBS, and acetylated in 0.5% acetic anhydride in 0.1 M Tris–HCl buffer, pH 8.0 for 10 min in Coplin jars on a shaker, washed (3⫻5 min) with TBS, treated with proteinase K (10 ␮g/ml) at 37 °C for 30 min, washed with TBS (3⫻5 min room temperature (RT), 1⫻5 min 4 °C), treated with 0.1% Triton X-100 in TBS (1⫻10 min), washed with TBS (1⫻5 min), dehydrated through a graded series of alcohols (70%, 95% and 100%), delipidated in chloroform for 5 min, rinsed in absolute ethanol, and air-dried. DIG-labeled cRNA probes were diluted (1 ␮g/ml) in hybridization buffer [50% formamide, 10% dextran sulfate, 1⫻ Denhardt’s solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 0.5 M NaCl, 10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 0.5 mg/ml baker’s yeast RNA, 200 mM dithiothreitol]. Diluted probes (100 ␮l per section) were placed on each slide, and the sections were coverslipped. Slides were placed in plastic trays moistened with 5⫻ SSC (standard saline citrate buffer). Hybridization was performed in an incubator at 75 °C overnight. The following day, coverslips were lifted with 5⫻ SSC at 55 °C and sections were washed with 2⫻ SSC (4⫻30 min) at 55 °C, in Coplin jars on a shaker. Subsequently, sections were treated with RNAase (20 ␮g/ml) (30 min, 37 °C), washed with 2⫻ SSC (3⫻5 min, RT), with 0.1⫻ SSC (2⫻15 min at 60 °C and 2⫻15 min at RT) in Coplin jars on a shaker and finally with TBS pH 7.6 (2⫻5 min, RT). Immunohistochemical detection of the DIG-labeled riboprobes was carried out as follows: Sections were incubated in

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TBS pH 7.6 containing 0.3% Triton X-100 and 1% NGS for 40 min at RT. Afterward, sections were incubated overnight at 4 °C with goat alkaline-phosphatase-conjugated anti-digoxigenin-F(ab) fragments (1:500, Roche Diagnostics GmbH). Following antibody incubation, slides were washed in TBS pH 7.6 (3⫻10 min), and twice in an alkaline buffer containing 0.1 M Tris–HCl pH 9, 50 mM MgCl2 and 0.1 M NaCl. Alkaline phosphatase activity was developed by incubating the sections overnight at RT with the chromogen [2.625 mg nitro blue tetrazolium, 1.75 mg bromochloroindolyl phosphate (Roche Diagnostrics GmbH) and 2.4 mg levamisole diluted in 10 ml of alkaline buffer]. The enzymatic reaction was terminated by extensive rinsing in Tris–HCl 10 mM, pH 7.4 containing 1 mM EDTA. The specificity of in situ hybridization was assessed by hybridization with a sense probe, which did not lead to any detectable signal. In each assay brain sections from all eight categories of animals were processed concurrently.

5-HT1A immunohistochemistry Sections were thawed, post-fixed with ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer for 1 h, washed in phosphatebuffered saline (PBS) (3⫻5 min) and in PBS containing 0.4% Triton X-100 (3⫻5 min) and incubated in PBS containing 0.4% Triton X-100 and 10% normal goat serum (NGS) (DakoCytomation, Denmark) for 1 h at RT. Afterward, sections were incubated overnight at 4 °C with the rabbit polyclonal anti-5-HT1A antibody (Santa Cruz, USA) at a concentration of 2 ␮g/ml diluted in PBS containing 0.4% Triton X-100 and 4% NGS. Following primary antibody incubation, slides were washed in PBS (3⫻5 min) and incubated for 1 h at RT with a biotinylated goat anti-rabbit (DakoCytomation) at a concentration of 5 ␮g/ml diluted in PBS containing 2% NGS. Following three rinses in PBS (3⫻5 min), slides were exposed to the ABC reagent (DakoCytomation) for 60 min at RT. Slides were then washed in PBS (3⫻5 min) and stained with 3,3=-diaminobenzidine (DAB) (1.7 mM, Sigma-Aldrich, USA) diluted in Tris–HCl buffer (10 mM, pH 7.6) and 0.03% H2O2 for 2–5 min at RT. Finally, they were washed, dehydrated and coverslipped with DePex (SERVA, Germany) and analyzed microscopically under a bright-field microscope. In each assay brain sections from all eight categories of animals were processed concurrently.

5-HT1A receptor autoradiography Kinetic analysis of 5-HT1A receptors was performed using [3H]8hydroxy-2(di-n-propylamino)tetralin ([3H]-8-OH-DPAT, 129 Ci/mmol) (from New England Nuclear, USA), as ligand. Non-specific binding was determined in the presence of 10 ␮M 5-HT. Sections were pre-incubated in 170 mM Tris–HCl, 4 mM CaCl2, pH 7.6 buffer (Tris-buffer), at RT for 30 min and incubated with [3H]-8-OH-DPAT (RT, 4 h) at a concentration range (0.2–20 nM) in Tris-buffer, containing 0.01% L-ascorbic acid, 10 ␮M pargyline and 10 ␮M fluoxetine. Free radioligand was removed by washing in 4 °C Tris-buffer (2⫻5 min). Sections were finally dipped in ddH2O and dried with a stream of cold air. Tritium-sensitive film (MS-I Kodak, USA) was exposed to labeled dried sections, along with plastic [3H]standards (American Radiolabeled Chemicals Inc., USA) for 1 month, developed in Kodak D-19, fixed with Agfa Acidofix and cleared in running water. Quantitative image analysis of the autoradiographs was performed using SCION-Image software. Specific binding, ⬎95% of the total binding, was expressed as fmol/mg tissue. Values for the equilibrium dissociation constant (Kd) and the maximum binding sites (Bmax) for [3H]-8-OH-DPAT were determined using EBDA-LIGAND (BIOSOFT, USA), an iterative fitting program based on a one-site binding model. In each assay brain sections from all eight categories of animals were processed concurrently.

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Quantification of in situ hybridization and immunohistochemistry The image analysis program “Image Pro Plus” (Media Cybernetics, USA) was used for counting cells (A) positive for 5-HT1A mRNA or (B) positive for 5-HT1A protein, in four hippocampal areas: CA1, CA3, CA4 and dentate gyrus (DG). Cell counting was performed “blindly” by two independent investigators. A “threshold” was set in the image analysis system, in order to include in the counting only cells stained above a certain, pre-set degree. Four randomly selected sections from each brain (between lateral positions 0.90 and 1.90 according to the anatomical atlas of Paxinos and Watson, 1986) were evaluated and in each brain section cells were counted in three to six different optical fields of each hippocampal area investigated. For each animal, the number of positive cells per optical field was the average value calculated from the data from all optical fields in all brain sections evaluated.

Statistical analysis Data were analyzed by two way analysis of variance (ANOVA) with handling and sex as independent factors. For the comparison of 5-HT1A Kd values at the two different ages (PND30 and PND90), three way ANOVA were performed with “handling,” sex and age as independent factors. When interactions between the independent factors were detected, separate one way ANOVAs followed by Bonferroni post hoc tests were performed in order to identify specific differences between groups. The level of statistical significance was set at 0.05. All tests were performed with the SPSS software (Release 10.0.1, SPSS, USA).

Photomicrograph production For in situ hybridization and immunohistochemistry, high-resolution microscopic images were digitally captured using a digital CCD color video-camera (TK-C1381, JVC, Japan) on an optical microscope (Eclipse E400, Nikon, Japan) connected to a PC via an Image Pro Plus frame grabber (Image Pro Plus, Media Cybernetics, USA). For 5-HT1A receptor binding, high-resolution images were produced from autoradiographic films scanned using a CanoScan 8000F scanner (Canon, China). Composite photomicrographs were prepared with the Adobe Photoshop 7.0 (Adobe Systems, USA).

RESULTS 5-HT1A receptors Thirty day old animals (PND30). Effects of sex. No sex difference was observed in the number of cells positive for 5-HT1A mRNA or protein or in the number of 8-OHDPAT binding sites in any of the hippocampal areas examined. Effects of handling. Handling resulted in statistically significant decreased numbers of cells positive for 5-HT1A either mRNA or protein in the CA4 (F1,23⫽5.053, P⫽0.044 for the mRNA; F1,23⫽6.644, P⫽0.024 for the protein) and DG (F1,23⫽66.758, P⬍0.001 for the mRNA; F1,23⫽ 112.022, P⬍0.001 for the protein) of both males and females at the age of 30 days (Fig. 1). Moreover, the number of 8-OH-DPAT binding sites was also decreased in the CA4 and DG of handled animals of both sexes, compared with the respective non-handled (main effect of handling: F1,23⫽12.572, P⫽0.002 for CA4; F1,23⫽18.652, P⬍0.001 for DG) (Fig. 3). On the contrary, the affinity of 8-OH-DPAT binding sites (Kd) was not affected by handling in any hippocampal area, of animals of either sex.

Ninety day old animals (PND90). Effects of sex. The number of 5-HT1A mRNA positive cells was higher in female animals compared with males in the CA1 and DG (F1,23⫽123.422, P⬍0.001 for CA1; F1,23⫽125.982, P⬍0.001 for DG) (Fig. 1). Additionally, the number of 5-HT1A immunopositive cells was higher in female animals compared with males in the CA1 (F1,23⫽80.497, P⬍0.001), while in the CA4 (F1,23⫽17.320, P⫽0.001, main effect of sex) and DG (F1,23⫽109.622, P⬍0.001, main effect of sex) this sex difference was observed only in the non-handled animals (Fig. 1). Likewise, the number of 8-OH-DPAT binding sites was higher in female than male animals in the CA1 (F1,23⫽89.668, P⬍0.001), while in the CA4 (F1,23⫽4.811, P⫽0.040 for CA4, main effect of sex) and DG (F1,23⫽14.556, P⫽0.001, main effect of sex) this sex difference was observed only in the non-handled animals (Fig. 3). Handling effects. Two-way ANOVA on the data regarding the 90 day old animals revealed a significant handling⫻sex interaction for the CA1 area (F1,23⫽34.258, P⬍0.001), the CA4 (F1,23⫽19.674, P⬍0.001) and the DG (F1,23⫽41.077, P⬍0.001). More specifically, the number of 5-HT1A mRNA positive cells was increased as a result of handling in the CA1 (P⬍0.001, post hoc test), CA4 (P⬍0.04, post hoc test) and DG (P⬍0.001, post hoc test) of male animals, while it was decreased only in the CA4 of handled females (P⬍0.01, post hoc test), compared with the respective non-handled animals (Figs. 1 and 2). A similar statistical analysis on the data for the number of 5-HT1A immunopositive cells showed a significant handling⫻sex interaction (F1,23⫽27.391, P⬍0.001 for CA1; F1,23⫽24.674, P⬍0.001 for CA4; F1,23⫽259.459, P⬍0.001 for DG). The number of 5-HT1A immunopositive cells was increased in the CA1 (P⬍0.01, post hoc test), CA4 (P⬍0.01, post hoc test) and DG (P⬍0.001, post hoc test) of male handled animals, whereas it was decreased in these same brain areas (CA1, P⬍0.02; CA4, P⬍0.04 and DG, P⬍0.001 post hoc tests) in the handled females, compared with the respective non-handled (Figs. 1 and 2). A significant handling⫻sex interaction (F1,23⫽25.754, P⬍0.001 for CA1; F1,23⫽20.222, P⬍0.001 for CA4; F1,23⫽36.981, P⬍0.001 for DG) was also revealed regarding the number of 8-OH-DPAT binding sites. The number of 5-HT1A binding sites was increased in the CA1 (P⬍0.005, post hoc test), CA4 (P⬍0.05, post hoc test) and DG (P⬍0.04, post hoc test) of male handled animals, while it was decreased in these same brain areas (CA1, P⬍0.03; CA4, P⬍0.02 and DG, P⬍0.001 post hoc tests) in the handled females, compared with the respective non-handled (Fig. 3). No sex difference in 5-HT1A receptor levels was observed among handled animals, due to the handling-induced increase in the males and decrease in the females. More specifically, there was no statistically significant difference between handled males and females in the number of 5-HT1A immunopositive cells in the CA1, CA4 and DG or binding sites in CA4 and DG; in the CA1 handled females had higher number of 8-OH-DPAT binding sites than the handled males (P⬍0.04, post hoc test).

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Fig. 1. Bar graphs showing the effect of neonatal handling on 5-HT1A mRNA and protein levels in the different areas of the hippocampus of pre-pubertal (PND30) and adult (PND90) male and female rats. Bars represent means⫾S.D. n⫽6 For all groups of animals. * Statistically significant effect of handling, P⬍0.05; † statistically significant effect of sex, Pⱕ0.001; # statistically significant handling⫻sex interaction, P⬍0.001.

In contrast to the total number of 8-OH-DPAT binding sites (Bmax), their affinity (Kd) was not affected by handling in any hippocampal area, of animals of either sex. Visual inspection of the 5-HT1A receptor Kd values in the two age groups indicated that they were different. We thus proceeded to perform a three way ANOVA with sex, handling and age as independent factors for each of the three hippocampal areas (CA1, CA4 and DG). This statistical analysis yielded only a significant effect of age (F1,47⫽ 64.961, P⬍0.001 for CA1; F1,47⫽99.964, P⬍0.001 for CA4 and F1,47⫽97.194, P⬍0.001 for DG). These results confirmed that the 5-HT1A receptor had a smaller Kd (1.00 nM for CA1, 0.98 nM for CA4 and 1.02 nM for DG) in the 30

day old animals than in the 90 day old (1.43 nM for CA1, 1.60 nM for CA4 and 1.63 nM for DG).

DISCUSSION The early experience of neonatal handling altered, in an age-dependent manner, the expression of the 5-HT1A receptor gene in particular subregions of the hippocampus, resulting in different levels of the respective mRNA, protein, as well as number of binding sites in the handled compared with the non-handled animals, of both sexes. More specifically, among the young (30 day old) animals, those exposed to handling, both females and males, had

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Fig. 2. Neonatal handling results in increased 5-HT1A mRNA and protein in the CA1, and CA4 subfields and the DG of the hippocampus of adult male rats. Photomicrographs depicting results of in situ hybridization (for mRNA) and immunohistochemistry (protein). The panel on the top shows a Nissl-stained section indicating the different areas of the rat hippocampus.

lower number of cells positive for 5-HT1A mRNA or protein and fewer 5-HT binding sites. On the other hand, when adult (90 day old), handled males had higher numbers of cells positive for 5-HT1A mRNA or protein and more 5-HT binding sites than the non-handled, while the opposite held true in the females. In most cases there were parallel changes in mRNA, protein levels and number of binding sites. An exception to this was the group of adult females, where handling was shown to result in decreased 5-HT1A protein levels and binding capacity, but appeared to have no effect on the respective mRNA levels. Similar results showing discrepancies in the changes between the mRNA and the protein of the 5-HT1A receptor have been reported by others (Miquel et al., 1991; Chalmers et al., 1993; Zhang et al., 1999), and can be ascribed to differences in post-transcriptional modifications leading to altered stability of the mRNA and its processing, as well as different rates of translation and/or degradation of the receptor. In the adult animals females had higher 5-HT1A receptor levels than the males, a sex difference that is probably

the result of the activational effects of the gonadal hormones, since it was not observed in the young, pre-pubertal animals. Higher 5-HT1A receptor levels in the females than the males have also been reported by others: Zhang et al. (1999) have shown that 5-HT1A mRNA was significantly higher in female than in male rats in hippocampal subregions CA3, CA4, and the DG. Similarly, binding to the 5-HT1A receptor was shown to be higher in the CA1 region of the hippocampus of female, compared with male rats (Mendelson and McEwen, 1991). Interestingly, the human female brain has also been shown to have a higher 5-HT1A binding capacity than the male (Parsey et al., 2002). It should be pointed out however that the factors underlying the sex difference in 5-HT1A receptor levels remain unclear. Although sex steroids are known to modulate 5-HT1A binding (Fischette et al., 1983) and it has been suggested that estradiol increases 5-HT1A binding (Biegon et al., 1982; Biegon and McEwen, 1982), others have reported that estrogens do not alter the density of 5-HT1A receptors (Frankfurt et al., 1994). Most recently

A. Stamatakis et al. / Neuroscience 140 (2006) 1–11

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Fig. 3. Effects of neonatal handling on 5-HT1A binding in the hippocampus of pre-pubertal (PND30) and adult (PND90) male and female rats. (A) Autoradiographs of in vitro binding to the hippocampus of adult male rats. (B) Bar graphs showing the number of 5-HT1A binding sites in the different areas of the hippocampus of pre-pubertal and adult male and female rats. Bars represent means⫾S.D. n⫽6 For all groups of animals. * Statistically significant effect of handling, Pⱕ0.002; † statistically significant effect of sex, Pⱕ0.04; # statistically significant handling⫻sex interaction, P⬍0.001.

available data (Le Saux and Di Paolo, 2005) indicate that in the hippocampus the effects of estrogens on 5-HT1A receptors involve alterations in the signal transduction pathway activated by the 5-HT1A receptor. Three out of the four categories of handled animals (young males and females, and adult females) had lower 5-HT1A receptor levels in the hippocampus, than the respective non-handled. The lower levels of hippocampal 5-HT1A receptors of the handled animals could be related to their lower HPA axis reactivity, which is well known to be a consequence of neonatal handling (Levine, 1957; Ader and Grota, 1969; Hess et al., 1969). It should be noted that 5-HT1A receptor activation leads to HPA axis disinhibition, since these receptors are hyperpolarizing (Hoyer et al.,

2002), having an inhibitory action on hippocampal activity, which exerts a suppressing effect on HPA axis function (De Kloet et al., 1998). Thus less 5-HT1A receptors would mean more hippocampal inhibitory control, resulting in a less reactive HPA axis. The only category of handled animals which had higher levels of 5-HT1A receptors than the non-handled, were the adult males, a finding for which it is difficult to find a clear explanation. A plausible mechanism could involve the actions of testosterone (whose levels are higher in the adult than the pre-pubertal animals), which has been shown to suppress 5-HT1A receptor levels (Zhang et al., 1999) and the high levels of corticosterone, that male non-handled animals are known to have (Smythe et al., 1996; Papaioannou et al., 2002b;

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Panagiotaropoulos et al., 2004a). It is well documented that corticosterone represses 5-HT1A receptor gene transcription acting through both MR and GR (Meijer et al., 2000; Wissink et al., 2000; Ou et al., 2001). In adult animals exposure to a variety of chronic stress paradigms results in down-regulation of hippocampal 5-HT1A receptors. However when rats were exposed during postnatal development to the stressful stimulus of 24 h of maternal deprivation an increase in 5-HT1A receptors was observed on the next day, only in the CA1 region of the developing hippocampus (Vazquez et al., 2000). In contrast, in the long-term experimental paradigm of 30 days of environmental enrichment an increase in 5-HT1A receptors was found in all CA hippocampal fields of adult male rats (Rasmuson et al., 1998), in agreement with our results. It should be noted that environmental enrichment has been shown to have similar results as neonatal handling on adult HPA axis reactivity. On the other hand, prenatal stress, which has the opposite effects, results in decreased 5-HT1A receptor binding (Griffin et al., 2005). In the young, pre-pubertal animals handling resulted in decreased levels of 5-HT1A receptors. This change in the serotonergic system is accompanied, as previously shown, by handling-induced increased 5-HT levels (Papaioannou et al., 2002a), indicating that the alterations in the receptors could be compensatory, resulting in a serotonergic system in the pre-pubertal rat brain which functionally is similar to that of the non-handled. It is worth mentioning that when pubertal animals were subjected to the challenge of chronic stress (15 consecutive days of 5 min daily forced swimming) no differences between handled and non-handled animals in immobility times or circulating corticosterone levels were observed (data not shown). In contrast, neonatal handling had a sexually dimorphic effect on 5-HT1A receptor levels in the hippocampus of adult animals, leading to increased levels in the males and decreased in the females. The handlinginduced increase in the males and decrease in the females results in comparable 5-HT1A receptor levels in the two sexes. However, it has been well documented that females (irrespective of neonatal handling) have a higher 5-HT turnover than males (Carlsson and Carlsson, 1988; Papaioannou et al., 2002a). Thus the handling-induced decrease in 5-HT1A receptors in the adult females occurs in a background of decreased 5-HT availability and could contribute to an increased predisposition toward “depressive” behavior when challenged with chronic stressful stimuli. Interestingly, the pattern of the handling-induced changes in 5-HT1A receptor levels in the adult animals reflects the sex-dependent effect of handling on immobility times following chronic forced swimming, previously reported using animals of the same age and “handled” with the same experimental protocol: in the males, it is the handled, which have shorter immobility times, while in the females, the non-handled (Papaioannou et al., 2002b). It should be noted that immobility time in the forced swimming is considered an expression of “depressive” behavior (Porsolt et al., 1977) and there is evidence that 5-HT1A receptors are involved in animal models of depression (Overstreet et al., 2003; Froger et al., 2004). Furthermore,

it has been shown that depression is associated with low levels of 5-HT1A receptor mRNA in the hippocampus and the dorsolateral prefrontal cortex (Lopez-Figueroa et al., 2004) and binding sites in the mesiotemporal, occipital and parietal cortex of the human brain (Drevets et al., 2000). Immobility time in the forced swimming is also thought of as an index of a passive coping strategy adopted in order to deal with stressful stimuli (Borsini and Meli, 1988). Interestingly, it has been reported that low levels of 5-HT1A receptors are associated with passive coping strategies expressed either as a result of genetic constitution (Veenema et al., 2004), or early environmental manipulations, such as exposure to increased corticosterone levels through the maternal milk (Meerlo et al., 2001). The present study supports and extends previous results from both our laboratory and others (FernandezTeruel et al., 2002) showing that neonatal handling affects the development and function of the serotonergic system. In pre-pubertal handled animals, males and females, an increase in 5-HT levels and a decrease in its turnover were observed in various brain areas including the hippocampus (Papaioannou et al., 2002a). The serotonergic system appears to be sensitive to both the organizational actions of testosterone and the effects of handling, since handled, neonatally masculinized females had higher 5-HT levels and decreased turnover in the hippocampus, hypothalamus and striatum following repeated forced swimming stress, compared with normal females (Panagiotaropoulos et al., 2004b). In addition, neonatal handling has been shown to increase 5-HT2 receptor binding (Smythe et al., 1994b) and type 1A receptor sensitivity (Papaioannou et al., 2002b). We now report that neonatal handling can have a sexually dimorphic effect on 5-HT1A receptor levels in the hippocampus of adult animals, leading to increased levels in the males and decreased in the females. It is worth mentioning that neonatal handling has a similar sexually dimorphic effect on brain-derived neurotrophic factor (BDNF) levels. Among males, the handled have increased BDNF levels in the CA4 area of the hippocampus (Garoflos et al., 2005), while in the females there was no handlinginduced effect (F. Stylianopoulou, unpublished observations). BDNF has been shown to play an important role in the development and functioning of the serotonergic system (Mattson et al., 2004). It is interesting to note that, in addition to neonatal handling, whose effects we report here, other models of early experiences such as rearing in either an enriched environment (Rasmuson et al., 1998), or isolation (Preece et al., 2004), overcrowded housing during postnatal development (Daniels et al., 2000), nursing by mothers receiving corticosterone through the drinking water (Meerlo et al., 2001), prenatal stress (Griffin et al., 2005), as well as maternal deprivation (Vazquez et al., 2000) alter 5-HT1A receptor levels in the hippocampus. Furthermore, it has been shown that the 5-HT1A receptor is not only present, but functional during rat brain development (Lauder et al., 2000), and is believed to participate in the morphogenetic actions of 5-HT (Sikich et al., 1990; Riad et al., 1994). It is thus possible that 5-HT1A receptors play a significant role

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in the cellular mechanisms through which early experiences “imprint” their effects on the brain. Acknowledgments—This work was funded by the special account for research of the University of Athens (Greece). A.S. was supported by a fellowship from the Academy of Athens, Greece.

REFERENCES Ader R, Grota LJ (1969) Effects of early experience on adrenocortical reactivity. Physiol Behav 4:303–305. Angold A, Costello EJ, Erkanli A, Worthman CM (1999) Pubertal changes in hormone levels and depression in girls. Psychol Med 29:1043–1053. Antonijevic IA (2006) Depressive disorders: is it time to endorse different pathophysiologies? Psychoneuroendocrinology 31:1–15. Biegon A, Fischette CT, Rainbow TC, McEwen BS (1982) Serotonin receptor modulation by estrogen in discrete brain nuclei. Neuroendocrinology 35:287–291. Biegon A, McEwen BS (1982) Modulation by estradiol of serotonin receptors in brain. J Neurosci 2:199 –205. Birmaher B, Ryan ND, Williamson DE, Brent DA, Kaufman J, Dahl RE, Perel J, Nelson B (1996) Childhood and adolescent depression: a review of the past 10 years. Part I. J Am Acad Child Adolesc Psychiatry 35:1427–1439. Blier P, Ward NM (2003) Is there a role for 5-HT1A agonists in the treatment of depression? Biol Psychiatry 53:193–203. Borsini F, Meli A (1988) Is the forced swimming test a suitable model for revealing antidepressant activity? Psychopharmacology 94:147– 160. Bowen DM, Najlerahim A, Procter AW, Francis PT, Murphy E (1989) Circumscribed changes of the cerebral cortex in neuropsychiatric disorders of late life. Proc Natl Acad Sci U S A 86:9504 –9508. Breitschopf H, Suchanek G (1996) Detection of mRNA on paraffin embedded material of the central nervous system with DIG-labeled RNA probes. In: Nonradioactive in situ hybridization application manual (Grünewald-Janho S, Keesey J, Leous M, van Miltenburg R, Schroeder C, eds), pp 136 –140. Mannheim, Germany: Biochemica, Boehringer Mannheim GmbH. Carlsson M, Carlsson A (1988) A regional study of sex differences in rat brain serotonin. Prog Neuropsychopharmacol Biol Psychiatry 12:53– 61. Chalmers DT, Kwak SP, Mansour A, Akil H, Watson SJ (1993) Corticosteroids regulate brain hippocampal 5-HT1A receptor mRNA expression. J Neurosci 13:914 –923. Champagne FA, Francis DD, Mar A, Meaney MJ (2003) Variations in maternal care in the rat as a mediating influence for the effects of environment on development. Physiol Behav 79:359 –371. Chaouloff F (2000) Serotonin, stress and corticoids. J Psychopharmacol 14:139 –151. Cheetham S, Crompton M, Katona C, Horton R (1990) Brain 5-HT1 binding sites in depressed suicides. Psychopharmacology 102:544– 548. Cowan PJ (2000) Psychopharmacology of 5-HT1A receptors. Nucl Med Biol 27:437– 439. Daniels WM, Pietersen CY, Carstens ME, Daya S, Stein D (2000) Overcrowding induces anxiety and causes loss of serotonin 5HT-1a receptors in rats. Metab Brain Dis 15:287–295. De Kloet ER, Vreugdenhil E, Oitzl MS, Joels M (1998) Brain corticosteroid receptor balance in health and disease. Endocr Rev 19: 269 –301. Drevets WC, Frank E, Price JC, Kupfer DJ, Greer PJ, Mathis C (2000) Serotonin type-1A receptor imaging in depression. Nucl Med Biol 27:499 –507. Drevets WC, Frank E, Price JC, Kupfer DJ, Holt D, Greer PJ, Huang Y, Gautier C, Mathis C (1999) PET Imaging of serotonin 1A receptor binding in depression. Biol Psychiatry 46:1375–1387.

9

Drossopoulou G, Antoniou K, Kitraki E, Papathanasiou G, Papalexi E, Dalla C, Papadopoulou-Daifoti Z (2004) Sex differences in behavioral, neurochemical and neuroendocrine effects induced by the forced swim test in rats. Neuroscience 126:849 – 857. Fernandez-Teruel A, Gimenez-Llort L, Escorihuela RM, Gil L, Aguilar R, Steimer T, Tobena A (2002) Early-life handling stimulation and environmental enrichment. Are some of their effects mediated by similar neural mechanisms? Pharmacol Biochem Behav 73:233– 245. Fischette CT, Biegon A, McEwen BS (1983) Sex differences in serotonin 1 receptor binding in rat brain. Science 222:333–335. Frankfurt M, McKittrick CR, Mendelson SD, McEwen BS (1994) Effect of 5,7-dihydroxytryptamine, ovariectomy and gonadal steroids on serotonin receptor binding in rat brain. Neuroendocrinology 59: 245–250. Froger N, Palazzo E, Boni C, Hanoun N, Saurini F, Joubert C, DutriezCasteloot I, Enache M, Maccari S, Barden N, Cohen-Salmon C, Hamon M, Lanfumey L (2004) Neurochemical and behavioral alterations in glucocorticoid receptor-impaired transgenic mice after chronic mild stress. J Neurosci 24:2787–2796. Garoflos E, Panagiotaropoulos T, Pondiki S, Stamatakis A, Philippidis E, Stylianopoulou F (2005) Cellular mechanisms underlying the effects of an early experience on cognitive abilities and affective states. Ann Gen Psychiatry 4:8 –19. Gold PW, Chrousos GP (2002) Organization of the stress system and its dysregulation in melancholic and atypical depression: high vs low CRH/NE states. Mol Psychiatry 7:254 –275. Griffin WC 3rd, Skinner HD, Birkle DL (2005) Prenatal stress influences 8-OH-DPAT modulated startle responding and [(3)H]-8-OHDPAT binding in rats. Pharmacol Biochem Behav 81:601– 607. Hamet P, Tremblay J (2005) Genetics and genomics of depression. Metabolism 54(Suppl 1):10 –15. Hesen W, Joels M (1996) Modulation of 5-HT1A responsiveness in CA1 pyramidal neurons by in vivo activation of corticosteroid receptors. J Neuroendocrinol 8:433– 438. Hess JL, Denenberg VH, Zarrow MX, Pfeifer WD (1969) Modification of the corticosterone response curve as a function of handling in infancy. Physiol Behav 4:109 –112. Hoyer D, Hannon JP, Martin GR (2002) Molecular, pharmacological and functional diversity of 5-HT receptors. Pharmacol Biochem Behav 71:533–554. Joels M, De Kloet ER (1991) Effects of corticosteroid hormones on electrical activity in rat hippocampus. J Steroid Biochem Mol Biol 40:83– 86. Karten YJ, Nair SM, van Essen L, Sibug R, Joels M (1999) Long-term exposure to high corticosterone levels attenuates serotonin responses in rat hippocampal CA1 neurons. Proc Natl Acad Sci U S A 96:13456 –13461. Kennett GA, Chaouloff F, Marcou M, Curzon G (1986) Female rats are more vulnerable than males in an animal model of depression: the possible role of serotonin. Brain Res 382:416 – 421. Lauder JM, Liu J, Grayson DR (2000) In utero exposure to serotonergic drugs alters neonatal expression of 5-HT(1A) receptor transcripts: a quantitative RT-PCR study. Int J Dev Neurosci 28: 171–176. Le Saux M, Di Paolo T (2005) Changes in 5-HT1A receptor binding and G-protein activation in the rat brain after estrogen treatment: comparison with tamoxifen and raloxifene. J Psychiatry Neurosci 30:110 –117. Levine S (1957) Infantile experience and resistance to physiological stress. Science 126:405– 406. Levine S (1994) Maternal behavior as a mediator of pup adrenocortical function. Ann N Y Acad Sci 746:260 –275. Lopez JF, Chalmers DT, Little KY, Watson SJ (1998) Regulation of serotonin1A, glucocorticoid, and mineralocorticoid receptor in rat and human hippocampus: Implications for the neurobiology of depression. Biol Psychiatry 43:547–573.

10

A. Stamatakis et al. / Neuroscience 140 (2006) 1–11

Lopez JF, Liberzon I, Vazquez DM, Young EA, Watson SJ (1999) Serotonin 1A receptor messenger RNA regulation in the hippocampus after acute stress. Biol Psychiatry 45:934 –937. Lopez JF, Vazquez DM, Chalmers DT, Watson SJ (1997) Regulation of 5-HT receptors and the hypothalamic-pituitary-adrenal axis. Implications for the neurobiology of suicide. Ann N Y Acad Sci 836: 106 –134. Lopez-Figueroa AL, Norton CS, Lopez-Figueroa MO, Armellini-Dodel D, Burke S, Akil H, Lopez JF, Watson SJ (2004) Serotonin 5-HT1A, 5-HT1B, and 5-HT2A receptor mRNA expression in subjects with major depression, bipolar disorder, and schizophrenia. Biol Psychiatry 55:225–233. Mattson MP, Maudsley S, Martin B (2004) BDNF and 5-HT: a dynamic duo in age-related neuronal plasticity and neurodegenerative disorders. Trends Neurosci 27:589 –594. McKittrick CR, Blanchard DC, Blanchard RJ, McEwen BS, Sakai RR (1995) Serotonin receptor binding in a colony model of chronic social stress. Biol Psychiatry 37:383–393. Meaney MJ, Aitken DH, Viau V, Sharma S, Sarrieau A (1989) Neonatal handling alters adrenocortical negative feedback sensitivity and hippocampal type II glucocorticoid receptor binding in the rat. Neuroendocrinology 50:597– 604. Meaney MJ, Mitchell JB, Aitken DH, Bhatnagar S, Bodnoff SR, Iny LJ, Sarrieau A (1991) The effects of neonatal handling on the development of the adrenocortical response to stress: implications for neuropathology and cognitive deficits in later life. Psychoneuroendocrinology 16:85–103. Meerlo P, Horvath KM, Luiten PG, Angelucci L, Catalani A, Koolhaas JM (2001) Increased maternal corticosterone levels in rats: effects on brain 5-HT1A receptors and behavioral coping with stress in adult offspring. Behav Neurosci 115:1111–1117. Meijer OC, Williamson A, Dallman MF, Pearce D (2000) Transcriptional repression of the 5-HT1A receptor promoter by corticosterone via mineralocorticoid receptors depends on the cellular context. Neuroendocrinology 12:245–254. Mendelson SD, McEwen BS (1991) Autoradiographic analyses of the effects of restraint-induced stress on 5-HT1A, 5-HT1C and 5-HT2 receptors in the dorsal hippocampus of male and female rats. Neuroendocrinology 54:454 – 461. Miquel MC, Doucet E, Boni C, Mestikawy SE, Matthiessen L, Daval G, Verge D, Hamon M (1991) Central serotonin1A receptors: respective distributions of encoding mRNA, receptor protein and binding sites by in situ hybridization histochemistry, radioimmunohistochemistry and autoradiographic mapping in the rat brain. Neurochem Int 19:453– 465. Mitchell JB, Iny LJ, Meaney MJ (1990) The role of serotonin in the development and environmental regulation of type II corticosteroid receptor binding in rat hippocampus. Brain Res Dev Brain Res 55:231–235. Moore CL, Morelli GA (1979) Mother rats interact differently with male and female offspring. J Comp Physiol Psychol 93:677– 684. Murray CJ, Lopez AD (1996) Evidence based health policy: lessons from global burden of disease study. Science 274:740 –743. Noble RE (2005) Depression in women. Metabolism 54(Suppl 1): 49 –52. Nolen-Hoeksema S, Girgus JS (1994) The emergence of gender differences in depression during adolescence. Psychol Bull 115: 424 – 443. Ou XM, Storring JM, Kushwaha N, Albert PR (2001) Heterodimerization of mineralocorticoid receptors at a novel negative response element of the 5-HT1A receptor gene. J Biol Chem 276:14299 – 14307. Overstreet DH, Commissaris RC, De La Garza R 2nd, File SE, Knapp DJ, Seiden LS (2003) Involvement of 5-HT1A receptors in animal tests of anxiety and depression: evidence from genetic models. Stress 6:101–110. Panagiotaropoulos T, Papaioannou A, Pondiki S, Prokopiou A, Stylianopoulou F, Gerozissis K (2004a) Effects of neonatal han-

dling and sex on basal and chronic stress-induced corticosterone and leptin secretion. Neuroendocrinology 79:109 –118. Panagiotaropoulos T, Pondiki S, Papaioannou A, Alikaridis F, Stamatakis A, Gerozissis K, Stylianopoulou F (2004b) Neonatal handling and gender modulate brain monoamines and plasma corticosterone levels following repeated stressors in adulthood. Neuroendocrinology 80:181–191. Papaioannou A, Dafni U, Alikaridis F, Bolaris S, Stylianopoulou F (2002a) Effects of neonatal handling on basal and stress-induced monoamine levels in the male and female rat brain. Neuroscience 114:195–206. Papaioannou A, Gerozissis K, Prokopiou A, Bolaris S, Stylianopoulou F (2002b) Sex differences in the effects of neonatal handling on the animal’s response to stress and the vulnerability to depressive behaviour. Behav Brain Res 129:131–139. Park MK, Hoang TA, Belluzzi JD, Leslie FM (2003) Gender specific effect of neonatal handling on stress reactivity of adolescent rats. J Neuroendocrinol 15:289 –295. Parsey RV, Oquendo MA, Simpson NR, Ogden RT, Van Heertum R, Arango V, Mann JJ (2002) Effects of sex, age, and aggressive traits in man on brain serotonin 5-HT1A receptor binding potential measured by PET using [C-11]WAY-100635. Brain Res 954:173– 182. Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates, 2nd edition. San Diego: Academic Press. Porsolt RD, Le Pichon M, Jalfre M (1977) Depression: a new animal model sensitive to antidepressant treatments. Nature 266:730 – 732. Preece MA, Dalley JW, Theobald DE, Robbins TW, Reynolds GP (2004) Region specific changes in forebrain 5-hydroxytrypt amine1A and 5-hydroxytryptamine2A receptors in isolation-reared rats: an in vitro autoradiographic study. Neuroscience 123:725– 732. Rasmuson S, Olsson T, Henriksson BG, Kelly PA, Holmes MC, Seckl JR, Mohammed AH (1998) Environmental enrichment selectively increases 5-HT1A receptor mRNA expression and binding in the rat hippocampus. Mol Brain Res 53:285–290. Riad M, Emerit MB, Hamon M (1994) Neurotrophic effects of ipsapirone and other 5-HT1A receptor agonists on septal cholinergic neurons in culture. Dev Brain Res 82:245–258. Sikich L, Hickok JM, Todd RD (1990) 5-HT1A receptors control neurite branching during development. Dev Brain Res 56:269 –271. Smythe JW, McCormick CM, Meaney MJ (1996) Median eminence corticotrophin releasing hormone content following prenatal stress and neonatal handling. Brain Res Bull 40:195–199. Smythe JW, McCormick CM, Rochford J, Meaney MJ (1994a) The interaction between prenatal stress and neonatal handling on nociceptive response latencies in male and female rats. Physiol Behav 55:971–974. Smythe JW, Rowe WB, Meaney MJ (1994b) Neonatal handling alters serotonin (5-HT) turnover and 5-HT2 receptor binding in selected brain regions: relationship to the handling effect on glucocorticoid receptor expression. Dev Brain Res 80:183–189. Vallée M, Mayo W, Dellu F, Le Moal M, Simon H, Maccari S (1997) Prenatal stress induces high anxiety and postnatal handling induces low anxiety in adult offspring: correlation with stress-induced corticosterone secretion. J Neurosci 17:2626 –2636. Vazquez DM, Lopez JF, Van Hoers H, Watson SJ, Levine S (2000) Maternal deprivation regulates serotonin 1A and 2A receptors in the infant rat. Brain Res 855:76 – 82. Veenema AH, Koolhaas JM, de Kloet ER (2004) Basal and stressinduced differences in HPA axis, 5-HT responsiveness, and hippocampal cell proliferation in two mouse lines. Ann N Y Acad Sci 1018:255–265. Weaver IC, La Plante P, Weaver S, Parent A, Sharma S, Diorio J, Chapman KE, Seckl JR, Szyf M, Meaney MJ (2001) Early environmental regulation of hippocampal glucocorticoid receptor gene

A. Stamatakis et al. / Neuroscience 140 (2006) 1–11 expression: characterization of intracellular mediators and potential genomic target sites. Mol Cell Endocrinol 185:205–218. Wissink S, Meijer O, Pearce D, van Der Burg B, van Der Saag PT (2000) Regulation of the rat serotonin-1A receptor gene by corticosteroids. Biol Chem 275:1321–1326.

11

Young EA, Altemus M (2004) Puberty, ovarian steroids, and stress. Ann N Y Acad Sci 1021:124 –133. Zhang L, Ma W, Barker JL, Rubinow DR (1999) Sex differences in expression of serotonin receptors (subtypes 1A and 2A) in rat brain: a possible role of testosterone. Neuroscience 94:251–259.

(Accepted 27 January 2006) (Available online 13 March 2006)