Maternal separation alters serotonergic transporter densities and serotonergic 1A receptors in rat brain

Neuroscience 140 (2006) 355–365

MATERNAL SEPARATION ALTERS SEROTONERGIC TRANSPORTER DENSITIES AND SEROTONERGIC 1A RECEPTORS IN RAT BRAIN A. VICENTIC,* D. FRANCIS, M. MOFFETT, A. LAKATOS, G. ROGGE, G. W. HUBERT, J. HARLEY AND M. J. KUHAR

somedial nucleus of amygdala amygdaloid nuclei. Conclusions: Early life maternal separation and the extent of handling can alter adult brain serotonergic transporter and serotonergic 1A levels and function in the forebrain. Alterations in these serotonergic systems by early rearing conditions might increase vulnerability for behavioral disorders in adulthood. © 2006 Published by Elsevier Ltd on behalf of IBRO.

The Yerkes National Primate Research Center of Emory University, 954 North Gatewood Road, Atlanta, GA 30329, USA

Abstract—Rationale: The basic mechanisms underlying the association between early life maternal separation and adulthood psychiatric disorders are largely unknown. One possible candidate is the central serotonergic system, which is also abnormal in psychiatric illnesses. Neuroadaptational changes in serotonergic transporter and serotonergic 1A receptors may underlie links between early life stress and adulthood psychiatric disorders. Objective: The aim of this study was to investigate the consequences of a rat model of maternal separation on serotonergic transporter and serotonergic 1A receptor densities and function in adult rat forebrain. Methods: Rat pups were separated from dams from postnatal day 2 to postnatal day 14, each day, for zero time, 15 min and 180 min to determine the time-course of effects. A nonhandled group was added to control for the effects of handling by an experimenter compared with the animal facilityreared group. Quantitative [125I]3␤-(4-iodophenyl)tropan-2␤carboxylic acid methyl ester and [125I]-mPPI autoradiography was used to determine serotonergic transporter and serotonergic 1A densities, respectively. Adult rats were challenged with saline or serotonergic 1A agonist (ⴙ) 8-hydroxy-2-(di-npropylamino)tetralin, 0.4 mg/kg, s.c.) and plasma adrenocorticotropic hormone and corticosterone were determined. Results: serotonergic transporter and serotonergic 1A densities were significantly lower in the non-handled group in the paraventricular, arcuate, dorsomedial and ventromedial nuclei of the hypothalamus. The non-handled group also displayed lower serotonergic transporter and serotonergic 1A densities in the basolateral anterior, basolateral ventral and basomedial amygdaloid nuclei. Serotonergic transporter densities were also decreased in the CA3 area of the hippocampus in the non-handled group. In contrast, the maternal separation 15 min group displayed the highest serotonergic transporter and serotonergic 1A densities in the basomedial nucleus of amygdala, basolateral anterior nucleus of amygdala, basolateral ventral nucleus of amygdala and ba-

Key words: serotonin, early life stress, ACTH, corticosterone, affective disorders.

Maternal deprivation, neglect and physical and sexual abuse in early life have been linked to behavioral disorders in adulthood (Higley et al., 1996; Bremne and Vermetten, 2001; Heim and Nemeroff, 2001; Gartside et al., 2003; Daniels et al., 2004). These disorders include depression, anxiety, drug abuse (Hall, 1998; Arborelius et al., 1999; Sadowski et al., 1999; Anand and Scalzo, 2000), altered reproductive behavior (Greisen et al., 2005) and compromised learning (Zaharia et al., 1996; Huot et al., 2002). Studies of the neurobiological mechanisms underlying the interaction between early life adversities and adulthood mental disorders suggest involvement of multiple neurotransmitter systems (Heim et al., 2001). Much evidence indicates that changes in the activity of the central serotonin (5-HT) system plays a major role in many of these behavioral aberrations (Nemeroff et al., 1994; Baldwin and Rudge, 1995; Lopez et al., 1997). During the early postnatal period, 5-HT plays a fundamental role in the development of the CNS, and 5-HT neurotransmission is involved in the activation and feedback of hypothalamicpituitary-adrenal (HPA) axis throughout life. Because 5-HT action in the synapse is terminated by reuptake, the serotonin transporter (SERT) is critical for regulating serotonergic function. In human and non-human species, both stressful experiences and changes in the SERT gene or protein levels have been linked to behavioral disorders and drug abuse (Malison et al., 1998; Heinz et al., 2000; Huot et al., 2001). For example, genetic variants of the SERT gene have been associated with increased anxiety (Tjurmina et al., 2002; Ansorge et al., 2004), neuroticism and depressive symptoms while SERT knockout mice exhibit alterations in 5-HT1A receptors and a stress-mediated increase in corticosterone (CORT) response (Li et al., 1999). In humans, the risk for developing psychopathologies in the face of stressful events is more marked in those carrying a short allele of SERT gene that alters transporter transcription levels (Heils et al., 1997), activity (Stoltenberg et al., 2002) and density (Mellerup et al., 2001; Zalsman et al., 2005). Moreover, rhesus macaques with a short allele

*Corresponding author. Tel: ⫹1-404-727-8277; fax: ⫹1-404-727-3278. E-mail address: [email protected] (A. Vicentic). Abbreviations: ACTH, adrenocorticotropic hormone; AFR, animal facility rearing; ANOVA, analysis of variance; Arc, arcuate nucleus; BLA, basolateral anterior nucleus of amygdala; BLV, basolateral ventral nucleus of amygdala; BM, basomedial nucleus of amygdala; CORT, corticosterone; DM, dorsomedial nucleus; HPA, hypothalamic-pituitary-adrenal; MS, maternal separation; MS0, maternal separation zero time; MS15, maternal separation 15 min; MS180, maternal separation 180 min; NH, non-handled; PD, postnatal day; PVN, paraventricular nucleus; RIA, radioimmunoassay; RT, room temperature; RTIS5, 3␤-(4-iodophenyl)tropan-2␤-carboxylic acid methyl ester; SERT, serotonin transporter; SSRI, selective serotonin reuptake inhibitor; 5-HT, serotonergic/serotonin; 8-OH-DPAT, (⫹) 8-hydroxy-2-(di-n-propylamino)tetralin. 0306-4522/06$30.00⫹0.00 © 2006 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2006.02.008

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of SERT that were exposed to early life adversities exhibited augmented neuroendocrine responses to stress (Barr et al., 2004) suggesting that alterations in SERT may underlie a link between early life stress and adulthood psychopathologies. Alterations in SERT function can cause adaptive changes in 5-HT1A receptors (Hensler, 2002; Celada et al., 2004; Li et al., 2004). Desensitization of 5-HT1A receptors plays a role in therapeutic efficacy of selective serotonin reuptake inhibitors (SSRIs). In fact, several preclinical (Arborelius et al., 1996; Kinney et al., 2000; Dawson et al., 2002) and clinical studies (Maes et al., 1996; Sacristan et al., 2000) reported that combined administration of 5-HT1A antagonists with SSRIs produces an earlier therapeutic effect than SSRIs alone. In addition, changes in 5-HT1A receptor densities were found in depressed humans with decreased SERT levels (Kasper et al., 2002) while changes in 5-HT1A receptor function were noted in SERT knockout mice (Li et al., 2004). Moreover, early maternal separation (MS) also seems to have consequences on 5-HT1A receptor densities in Octodon degus (Ziabreva et al., 2003a,b) and on 5-HT1A mRNA in rat pups (Vazquez et al., 2000). Likewise, alterations in 5-HT1A receptor function were observed in adult rats exposed to a six hour perinatal MS (Gartside et al., 2003). However, not all studies of MS revealed changes. For example, one study found no changes in 5-HT1A mRNA in the dorsal raphe nucleus or in hippocampal area CA1 (Neumaier et al., 2002), while another found no changes in SERT or 5-HT1A mRNA or binding in the dorsal raphe nucleus, although a change in the sensitivity of citalopram on 5-HT firing rate was reported (Arborelius et al., 2004). Despite the inconsistent findings and limited number of brain regions examined, these prior results suggest that MS can produce changes in SERT and 5-HT1A systems that may persist into adulthood. Thus, given this previous evidence and suggestion that SERT and 5-HT1A receptors are important links between early life stress and adulthood behavioral disorders, further studies on the consequences of MS on neuroadaptive responses of SERT and 5-HT1A receptor are warranted. To date, no studies have investigated SERT and 5-HT1A receptor densities throughout the forebrain as a consequence of MS. Therefore, the first goal of this study was to investigate whether early in life MS or alterations in handling alter SERT and 5-HT1A receptor densities in discrete areas throughout the forebrain. Because changes in binding levels may not necessarily produce changes in function, another goal was to examine if the changes in 5-HT1A receptor binding produced functional changes by measuring agonist-induced hormonal responses that are mediated by these receptors. Finally, because of varying experimental conditions among different groups, the third goal was to use a rodent model of early MS that includes five groups of rats to control for duration of separation and experimenter handling, including an animal facility-reared group (AFR) (Jaworski et al., 2005). Groups of pups separated from dams for zero time (MS0), for 15 min (MS15) and for 180 min (MS180) al-

lowed obtaining relevant time-course data. Moreover, a non-handled group (NH) controlled for effects of handling by experimenter compared with that of the animal care takers (the AFR groups) without having separation as a variable. The hypotheses of these studies were as follows: (1) variations in MS and handling in the perinatal period will alter SERT and 5-HT1A receptors in adulthood; (2) these changes will be evident in many forebrain regions, and (3) changes in binding will have functional consequences. To our best knowledge, this is the first study that uses such an extensively controlled paradigm of early life MS to elucidate long-term effects on SERT and 5-HT1A receptor systems throughout the brain.

EXPERIMENTAL PROCEDURES Animals Timed-pregnant, female, Long Evans rats were purchased from Charles River Laboratories (Wilmington, MA, USA) and were singly housed in the same room maintained on a 12-h light/dark cycle (lights on at 7:00 a.m.). Cages were transparent with the following dimensions: 27 cm wide⫻48 cm deep⫻20 cm high. Cage bedding consisted of Bed-o’-cobs laboratory bedding (The Andersons, Maumee, OH, USA). Cages were placed on a static shelf for the duration of the experiment. All experiments were carried out according to the Principles of Laboratory Animal Care (http://www. nap.edu/readingroom/books/labrats) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the protocol was approved by the Emory University Institutional Animal Care and Use Committee. The approved experimental protocol employed a minimal number of animals with all necessary care taken to minimize any discomfort. A total of 30 litters were born and randomly assigned to one of five experimental groups (MS0, MS15, MS180, NH and AFRs). Since we had 30 litters in total, six litters were present in each of our five treatment groups. A majority of studies (Francis et al., 2002; Huot et al., 2002; Meaney et al., 2002; Arborelius et al., 2004) including ours (Jaworski et al., 2005) that have utilized a similar paradigm of MS employed male animals. To extend our previous observations in males (Jaworski et al., 2005) by studying the effects of MS on other neurobiological systems and mechanisms, we employed only male animals. Thus, one or two rats from each litter within the group were used in individual experiments in order to avoid “litter effect.” In other words, when eight rats within a given treatment (for example MS0) were needed for an experiment, rats were taken from each of the six litters within that treatment. We carried out four to five separate experiments and used five to eight animals from each litter. Therefore the unit of measure was a group of animals from six litters with each animal subjected to the same treatment. The litter sizes were purposely not culled. No significant difference in the average litter size was observed within each group. Likewise, one way analysis of variance (ANOVA) showed no significant differences in body weights per group at that time or at adulthood F(4,44)⫽0.74, P⫽0.57; data not shown). The statistical details of this paradigm were published in our previous study (Jaworski et al., 2005). Taken together, these results argue against a litter effect. The separation paradigm (postnatal day (PD) 2–14) for MS15 and MS180 was carried out as described previously (Jaworski et al., 2005). Briefly, the dam was first removed from the homecage and placed in a new cage. Subsequently, the pups were individually picked and removed from the homecage (for 15 min, MS15 or for 180 min, MS180), and then placed together in a different new cage for varying amounts of time. At the end of the separation time, all pups were returned in the homecage first and then the dam was replaced. In the MS0 group, there was touching of pups

A. Vicentic et al. / Neuroscience 140 (2006) 355–365 and dams but no separation from the cage; the pups were picked up one at a time and placed at opposite end of homecage, and then mother was picked up and placed at opposite end of cage. The homecage was then replaced on the rack and left undisturbed until the following day. All animals were handled with gloved hands. During the separations all mothers and pups were being touched and handled. To control for this effect we included another group, NH, in which the mothers and pups were only touched on PD 11 for cage change and not separated. In addition, a standard AFR group was also included. In this group, animals were transferred to new cages, (new bedding and water) twice per week as a part of the facility routine. Like the NH, the AFR animals were never separated but unlike the NH, their cages were changed four times during the experiment. At PD 21 male offspring were housed three per cage across experimental groups and subsequently tested in adulthood.

Drugs (⫹) 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and 5-HT were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Iodinated [125I]3␤-(4-iodophenyl)tropan-2␤-carboxylic acid methyl ester (RTI-55) and [125I]-MPPI [4-(2=methoxyphenyl)-1-[2=[2=-pyridinyl-)-iodo-benzamido]ethyl]piperazine were purchased from PerkinElmer Life Sciences Inc (Boston, MA, USA). Autoradiographic [125I]microscales were purchased from Amersham Bioscience (Piscataway, NJ, USA). Radioimmunoassay (RIA) kits for CORT and adrenocorticotropic hormone (ACTH) (Coat-a-Count) were purchased from Diagnostic Products (Los Angeles, CA, USA).

Autoradiographic studies The adult male rats were killed by decapitation. Whole brains (n⫽5) were removed and immediately frozen on dry ice and stored at ⫺80 °C. Fourteen micron thick coronal sections were obtained using a cryostat (Leica, CM3000; Leica Microsystems Inc., Bannockburn, IL, USA). Sections were thaw-mounted onto chromealuminum/gelatin-coated glass microscope slides and stored at ⫺20 °C until used for autoradiographic analysis of SERT and 5-HT1A receptor densities. Coronal sections were obtained at the following levels according to the rat atlas (Paxinos and Watson, 1986): from bregma, striatum (⫹1.00 to ⫺0.5 mm), hypothalamus, amygdala and hippocampus (⫺1.60 mm to ⫺3.40 mm). 125 I-MPPI-labeled 5-HT1A binding sites were determined by autoradiography as previously described (Kung et al., 1995; Li et al., 2000) with slight modifications. Briefly, the slides were thawed and dried in a desiccator at room temperature (RT). The brain sections were pre-incubated at RT for 30 min in assay buffer (50 mM Tris–HCl, pH⫽7.4, containing 2 mM MgCl2). Slides were incubated for 120 min in assay buffer containing 125I-MPPI at a final concentration of 0.14 nM. Non-specific binding was defined in the presence of 10 mM 5-HT. The slides were washed two times with ice-cold assay buffer for 15 min and then briefly rinsed in ice-cold ddH2O. After being blow dried the slides were left in a desiccator overnight prior to being apposed to Kodak, BiomaxMS01 film for 24 h. In order to convert the optic density readings into fmol/mg of tissue equivalent, a set of 125I standard microscales was apposed together with the slides. 125 I-RTI-55-labeled SERT binding sites were determined by autoradiography as previously described with slight modifications (Boja et al., 1992; McGregor et al., 2003). The slides were thawed and dried in a desiccator at RT. The brain sections were preequilibrated at RT for 30 min in phosphate buffer (10 mM NaH2PO4, 0.1 M sucrose, pH⫽7.4). SERT binding was defined by incubating slides for 120 min in assay buffer containing 50 pM 125 I-RTI-55 in the presence of 1 ␮M mazindol. Non-specific binding was defined in the presence of 50 nM citalopram. Following incubation, the slides were washed (1⫻1 min, then 2⫻20 min) in ice-cold buffer, followed by a brief dip in ice-cold ddH2O. The

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slides were dried under the gentle stream of dry air and dried overnight in a desiccator. The slides were apposed to Kodak, Biomax-MS01 film for 16 h in the presence of 125I standard microscales in order to calculate optical density readings into fmol/mg tissue equivalent.

Data analysis Autoradiographic film densities were quantified on a PC computer (Dell XPS H266) driven by the public domain Scion Image software (Scion Corporation, Frederick, MD, USA) based on the NIH Image program. The calculation of SERT and 5-HT1A receptor densities was based on the quantified amounts of radioactivity present in each layer of the polymer standards. Non-linear best-fit regression analysis was used to determine the relationship between the optical densities of the iodinated standards on film versus the known molar concentration of radioactivity contained in the different layer of the standards. Best-fit standard curves of film gray scale values generated by iodinated standards co-exposed with labeled slides resulted when an exponential function was used to describe the relationship between radioactive standards and gray scale value. For each brain region a mean determination of the fmol of sites per mg tissue equivalent in individual animals was carried out by averaging the values obtained from six to eight adjacent sections (measurements were performed from both left and right sides of the autoradiograms). These values represented the total signal. After averaging the left and right side, the mean total value of a particular section was obtained. The mean nonspecific binding was determined in four adjacent sections from each animal.

Pharmacological challenges Animals (n⫽8) were handled for four consecutive days before the experiment to minimize stress. Functional alterations in 5-HT1A receptors were determined on PD 80 by measuring the magnitude of elevation in plasma hormones following a single s.c. injection of either saline or the selective 5-HT1A receptor agonist (⫹)8-OH-DPAT (0.4 mg/kg). This dose of (⫹)8-OH-DPAT was shown to produce a maximal responses in our earlier studies (Vicentic et al., 1998; D’Souza et al., 2004). The animals were killed (between 10:00 a.m. and 12:00 p.m.) by decapitation 15 min post-injection, and trunk blood was collected in ice-cold centrifuge tubes containing 0.5 ml of a 0.3 M ethylenediamine tetraacetic acid (EDTA) (pH 7.4) solution. This post-injection time yields nearly peak responses for ACTH and robust, but less than peak increases in CORT. After centrifugation at 2500 r.p.m. at 4 °C for 30 min, plasma was aliquoted and stored at ⫺80 °C for subsequent radioimmunoassays of plasma CORT and ACTH.

RIA of CORT and ACTH Assays were performed in the Endocrine Core Laboratory at the Yerkes National Primate Research Center. Plasma CORT was determined by radioimmunoassay of 25 ␮l duplicates using commercially available RIA reagents. The assay has a sensitivity of 0.02 ␮g/dl and an intra- and interassay CV of 3.1% and 7.6%, respectively. Plasma ACTH was determined by radioimmunoassay of 100 ␮l duplicates using a commercially available kit (DiaSorin, Stillwater, MN, USA). The assay has a sensitivity of 4.5 pg/ml and an intra- and interassay CV of 7.1% and 12.4%, respectively.

RESULTS Five groups of rats were treated in the postnatal period as described in Experimental Procedures and then killed in adulthood. The brains were frozen, sectioned and the autoradiographic localization and quantification of SERT and

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Table 1. Quantitative autoradiography of SERT binding in adult animals exposed to AFR, MS0, MS15, MS180 and NH during PD2-PD14 Region Cortex Fr Cg1-2 Par-1 Tu Ent Nucleus accumbens Core Shell Hypothalamus PVN LH DM VMN Arc Amygdala BLA BLV BM Hippocampus CA1 CA2 CA3

MS0

MS15

MS180

NH

AFR

27⫾0.8 22⫾0.1 26⫾0.5 43⫾2.1 30⫾3.1

35⫾5.1 23⫾0.5 27⫾0.7 48⫾1.6 31⫾2.6

30⫾0.4 19⫾0.2 28⫾1.0 47⫾0.9 33⫾3.4

28⫾1.5 17⫾0.4 27⫾0.2 50⫾2.8 29⫾3.5

31⫾0.4 21⫾0.5 28⫾0.9 45⫾3.1 34⫾3.0

49⫾2.7 32⫾0.8

54⫾1.9 31⫾2.2

52⫾4.4 33⫾0.5

46⫾0.6 34⫾0.9

48⫾3.7 31⫾0.5

59⫾5.2 67⫾3.6 61⫾2.1 55⫾5.6 29⫾2.0

48⫾2.4 57⫾3.3 58⫾2.7 45⫾0.4 29⫾3.3

42⫾1.3 58⫾3.2 56⫾6.0 46⫾7.5 25⫾3.4

35⫾3.2* 47⫾3.6* 50⫾5.1# 39⫾3.4# 20⫾1.1*

51⫾1.1 68⫾2.3 73⫾4.6 59⫾9.8 35⫾0.3

160⫾2.4 129⫾5.8 120⫾7.5

180⫾4.0& 152⫾5.9& 134⫾2.4&

159⫾3.4 136⫾4.7 124⫾9.2

127⫾1.3* 100⫾2.3* 90⫾4.3†

152⫾2.3 127⫾2.0 115⫾1.2

24⫾2.5 28⫾1.2 33⫾1.7

20⫾1.4 30⫾3.2 28⫾1.4

27⫾1.3 24⫾1.0 28⫾2.1

20⫾0.7 25⫾0.8 24⫾2.5#

26⫾0.4 31⫾0.7 37⫾2.3

The data represent mean⫾S.E.M. from five animals. Abbreviations: CA1, field of Ammon’s horn; CA2, field of Ammon’s horn; CA3, field of Ammon’s horn; Cgl-2, cingulated cortex; Ent, enthorinal cortex; Fr, frontal cortex; LH, lateral hypothalamus; Nac, nucleus accumbens; Par1, parietal cortex area 1; PVN, paraventricular nucleus of the hypothalamus; Tu, olfactory tubercle; VMH, ventromedial hypothalamic nuclei. * Significantly different from AFR, MS0, MS15 and MS180 groups. # Significantly different from AFR group. † Significantly different from MS15 group. & Significantly different from NH and AFR group.

5-HT1A receptors were carried out as described in Experimental Procedures. After determining the binding distribution in many brain regions we observed that our results were generally comparable to those in previous reports (Boja et al., 1992; McGregor et al., 2003) and that changes were found in some areas. Several limbic and other regions were selected for a more complete analysis (Tables 1 and 2). A one-way ANOVA followed by a Newman-Keuls post hoc test revealed significant differences in SERT densities among the groups in specific areas of the hypothalamus, amygdala and hippocampus (Table 1). In general, SERT densities in NH animals were significantly lower than in all other treatment groups in the paraventricular nucleus (PVN) (F(4, 24)⫽18.1, P⬍0.0001), LH (F(4, 24)⫽16.1, P⬍0.0001) (Fig. 1) and arcuate nuclei (Arc) (F(4, 24)⫽8.5, P⬍0.001). SERT levels in the NH group were also lower than in the AFR group in dorsomedial nucleus (DM) (F(4, 24)⫽4.9, P⬍0.01) and in VMH (F(4, 24)⫽4.0, P⬍0.05) hypothalamic nuclei (Table 1). Similarly, SERT densities were decreased in discrete nuclei of the amygdala in the NH group, including basolateral anterior nucleus of amygdala (BLA) (F(4, 24)⫽ 28.7, P⬍0.0001) and basolateral ventral nucleus of amygdala (BLV) (F(4, 24)⫽29.8, P⬍0.0001) compared with all other groups, and in basomedial nucleus of amygdala (BM) (F(4, 24)⫽4.5, P⬍0.05) compared with MS15 group (Table 1 and Fig. 2.). Also in these amygdaloid nuclei, SERT densities were significantly higher in MS15 com-

pared with those in NH and AFR groups (F(4, 24)⫽4.8, P⬍0.05). In the hippocampus, SERT levels were significantly lower only in the CA3 area (F(4, 24)⫽10.6, P⬍0.0001) of NH animals compared with AFR. The [125I]-mPPI binding in discrete brain regions showed specific labeling of 5-HT1A receptors. The general regional distribution of [125I]-mPPI labeled 5-HT1A receptors matched those labeled by [125I]8-OH-DPAT and was in general agreement with previous studies (Kung et al., 1995; Li et al., 1999). The 5-HT1A receptor densities (Table 2, Fig. 3) were significantly different among the treated groups in discrete areas of the hypothalamus and amygdala. In the hypothalamus, a one-way ANOVA followed by a Newman-Keuls post hoc test revealed a significant decrease (F(4, 24)⫽13.8, P⬍0.0001) in 5-HT1A binding densities in the PVN and LH (Table 2, Fig. 3) of the NH group compared with all other treatment groups, and in the DM and VMH of the NH group compared with the AFR (Table 2). Attenuation of 5-HT1A densities was found also in the BLA, BLV and BM nuclei of the amygdala of the NH group (Table 2, Fig. 4). The 5-HT1A receptor levels in these amygdaloid nuclei were significantly higher (F(4, 24)⫽7.2, P⬍0.001) in the MS15 group compared with all other treatment groups. Because the above autoradiographic analysis indicated changes in 5-HT1A receptor densities, we tested the functional significance of these changes by measuring

A. Vicentic et al. / Neuroscience 140 (2006) 355–365

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Table 2. Quantitative autoradiography of 5-HT1A receptor binding in adult animals exposed to AFR, MS0, MS15, MS180 and NH during PD2-PD14 Region Cortex Fr I–IV Fr V–VI Par I–IV Par V Ent Nucleus accumbens Core Shell Hypothalamus PVN LH DMD VMH Arc Amygdala BLA BLV BM Hippocampus CA1 CA2 CA3

MS0

MS15

MS180

NH

AFR

42⫾5.4 84⫾15.8 93⫾11.1 125⫾21.6 100⫾13.9

40⫾3.8 89⫾10.6 90⫾15.9 116⫾21.5 96⫾15.7

39⫾2.7 91⫾8.4 101⫾13.4 120⫾18.6 95⫾8.9

35⫾3.0 87⫾12.1 100⫾10.9 123⫾8.9 88⫾10.9

38⫾3.2 103⫾10.8 92⫾8.7 132⫾32.2 94⫾11.5

32⫾5.3 34⫾0.9

37⫾1.9 31⫾2.2

32⫾6.2 33⫾0.5

34⫾1.7 34⫾0.9

36⫾1.8 30⫾0.5

48⫾6.5 33⫾3.4 74⫾4.5 60⫾2.8 19⫾2.2

42⫾1.8 30⫾1.3 72⫾1.9 52⫾3.4 22⫾0.5

39⫾2.5 26⫾3.3 65⫾6.8 56⫾2.9 21⫾0.5

29⫾0.8* 22⫾1.1* 58⫾2.1# 39⫾1.8# 17⫾6.3

40⫾1.0 35⫾1.9 79⫾0.5 66⫾2.6 24⫾5.1

56⫾2.1 39⫾1.8 126⫾7.9

64⫾1.1& 43⫾1.3& 132⫾4.8&

49⫾5.6 33⫾0.6 119⫾2.2

38⫾2.1* 28⫾1.2* 81⫾0.3*

51⫾2.1 35⫾1.7 111⫾2.5

231⫾17.7 80⫾4.2 102⫾2.7

250⫾15.6 86⫾2.5 90⫾5.3

238⫾22.1 78⫾2.0 92⫾5.3

222⫾22.8 79⫾2.0 84⫾2.2

246⫾6.8 82⫾4.5 94⫾0.7

The data represent mean⫾S.E.M. from five animals. Abbreviations: CA1, field of Ammon’s horn; CA2, field of Ammon’s horn; CA3, field of Ammon’s horn; Ent, enthorinal cortex; Fr, frontal cortex lamina I–IV and V–VI; LH, lateral hypothalamus; Par1, parietal cortex lamina I–IV and V–VI; VMH, ventromedial hypothalamic nuclei. * Significantly different compared to AFR, MS0, MS15 and MS180 group. # Significantly different from AFR group. & Significantly higher than AFR, MS0, MS180 and NH.

8-OH-DPAT-induced and 5-HT1A receptor-mediated changes in CORT and ACTH levels. Consistent with previous findings (Vicentic et al., 1998), 8-OH-DPAT (0.4 mg/kg, s.c.) significantly increased plasma CORT (Fig. 5A) and ACTH levels (Fig. 5B). A two-way ANOVA followed by a Newman-Keuls post hoc tests revealed that the 8-OH-DPATinduced increase in CORT and ACTH was significantly attenuated (F(4, 26)⫽9.9, P⬍0.0001) in NH animals compared with all other treatment groups, suggesting a blunted

functional response of 5-HT1A receptors to an agonist challenge. This finding further suggests that changes in 5-HT1A receptor densities reflect functional changes as well.

DISCUSSION The present study examined SERT levels and 5-HT1A receptor levels and function in the brains of adult rats

Fig. 1. Dark field autoradiograms of a typical MS15 and NH animal demonstrating [125I]RTI-55-labeled SERT densities in the PVN and LH nuclei of the hypothalamus. SERT densities were significantly (P⬍0.05) lower in these nuclei in the NH group. See text for more details.

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Fig. 2. Dark field autoradiograms of a typical MS15 and NH animal demonstrating [125I]RTI-55-labeled SERT densities in the Arc nucleus of the hypothalamus, and BLA, BLV and BM nuclei of the amygdala. SERT densities were significantly (P⬍0.05) lower in these nuclei in the NH group and significantly higher in the BLA, BLV and BM nuclei of amygdala in the MS15 group. See text for more details.

exposed to early life MS and variations in handling. It substantially extends our knowledge of separation-induced changes in SERT and 5-HT1A receptor densities across several regions of the rat brain, and provides several novel findings about how handling and MS can affect serotonergic systems in adulthood. The majority of changes were found in the hypothalamus and amygdala with the highest SERT and 5-HT1A densities in the MS15 group and lowest in the NH group. The NH group had almost no experimenter or caretaker handling during the two week postnatal period; they were handled only once for a required cage change (Jaworski et al., 2005). These results generally agree with our previous findings in an alcohol self-administration study (Jaworski et al., 2005) and suggest that handling is an important form of perinatal manipulations contributing to the changes. The finding that the NH group, in some studies regarded as a “control,” exhibited majority of changes, although more difficult to interpret, is not surprising. Recent

study demonstrated that the NH and MS180 animals exhibited a higher fear response in the open field test compared with animals handled for 15 min which is our MS15 group (Madruga et al., 2005). Similarly, Champagne and Meaney (2001), Francis et al. (2002), and Ruedi-Bettschen et al. (2005) showed that lack of handling (NH group) is linked to higher stress and anxiety compared with short separation of 15 min. In addition, compared with the MS15, both NH and MS180 animals have reduced GABA(A) receptor levels in the locus coeruleus and reduced central benzodiazepine receptor sites in the amygdala, a critical site for fear behaviors and the anxiolytic effects of benzodiazepines (Caldji et al., 2000b). Our preliminary results (not shown) in the open field test also suggest that MS15 group appears to be least anxious while the NH and MS180 groups have higher levels of anxiety. Furthermore, Brake et al. (2004) demonstrated that NH and MS180 group have higher vulnerability to stress compared with the MS15s. Surprisingly, despite the similar behavioral pheno-

Fig. 3. Dark field autoradiograms of a typical MS15 and NH animal demonstrating [125I]mPPI-labeled 5-HT1A receptor densities in the PVN and LH nuclei of the hypothalamus in adult rat brain. Significant (P⬍0.05) decreases in 5-HT1A receptor densities were detected in these nuclei in the NH group. See text for more details.

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Fig. 4. Dark field autoradiograms of a typical MS15 and NH animal demonstrating [125I]mPPI-labeled 5-HT1A densities in the Arc nucleus of the hypothalamus, and BLA, BLV and BM nuclei of the amygdala. SERT densities were significantly (P⬍0.05) lower in these nuclei in the NH group and significantly higher in the BLA, BLV and BM nuclei of amygdala in the MS15 group. See text for more details.

type, these two groups differed in their autoradiographic measurements of dopamine transporter and D3 receptor densities (Brake et al., 2004). In addition, compared with MS15, the MS180 did not consistently display decreases in these DA systems densities which suggests that despite its more “vulnerable” phenotype, neuroanatomical measurements do not necessarily parallel behavioral measurements, in this case the assessment of stress. Likewise, we also demonstrated that the NH and MS180 groups differed in their SERT and 5-HT1A receptor

Fig. 5. Effects of MS on 8-OH-DPAT-induced (0.4 mg/kg s.c.) stimulation on 5-HT1A receptor-mediated secretion of plasma CORT (A) and ACTH (B). * Indicates a significantly (P⬍0.05) lower 8-OH-DPATstimulated plasma CORT and ACTH levels in the NH group of animals.

densities in some brain areas. In addition, we showed that the MS15 exhibited higher 5-HT1A receptor densities in amygdala compared with all groups including the MS180 and MS0. Although the MS180, exhibited lower 5-HT system densities compared with MS15, the fact that other groups were also lower, suggests that this change cannot be simply related to time of separation. It also suggests that depending on the system examined, the NH condition represents a form of stress for animals. Thus, in the absence of any apparent postnatal perturbation, the observed findings in amygdala and hypothalamus cannot be simply related to time of separation but to more complex mechanisms that played a role in anatomical and functional differences between the groups. Another interesting finding relates to the AFR group which was not different from the MSO or the MS180 groups but it differed from the NH group. While the experimenter carried out and consistently controlled every detail of the experiment from the moment pups were born throughout the separation paradigm, the AFR group was cared by animal caregivers. Moreover, the AFR group had three cage changes compared with the NH which had only one at PD11. Thus, differences in rearing conditions of AFR compared with NH and other groups could have influenced their differences in the 5-HT measurements. It also underscores the importance of selecting more groups for comparison purposes. Moreover, due to differences in the basic animal care and handling (AFR) among facilities (Huot et al., 2001; Ploj et al., 2003), employing the AFR as a control may not the most favorable option although this group may provide some information as to the effects of “routine” animal care. Finding changes in some brain regions and not others indicates that not all 5HT neurons are affected in the same way by separation and handling. Indeed, opposite changes in the amygdala of NH and MS15 animals support this. Some investigators have proposed that the relative stress among the groups is the major factor in their responses (Francis et al., 1999; Caldji et al., 2000a; Meaney, 2001). Others suggest that the reactions of the dams to MS and

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handling are important and capable of producing changes in pups (Denenberg, 1999; Champagne and Meaney, 2001; Champagne et al., 2003; Kalinichev et al., 2003). While these processes are not clearly understood, it has been proposed that MS produces widespread changes in several neurotransmitter systems that regulate HPA axis including the 5-HT. Another interesting finding is the lack of differences between the MS0 and MS180 groups. The MS0 group has not been studied often, if ever. The absence of differences in SERT and 5-HT1A receptor densities between MS0 and MS180 suggests that the neurochemical and behavioral changes that occur probably involve handling and other factors. The understanding of consequences of MS on SERT and 5-HT1A receptor system has been rather limited. The conditions of MS and rearing that varied significantly among different laboratories contributed differences in findings. In addition, the NH and MSO groups were not consistently included in other related studies. For example, repeated, brief periods of MS in Octodon degus caused an increase in 5-HT1A binding in the cortex of female pups (Ziabreva et al., 2003b). Moreover, an increase in 5-HT1A mRNA (Vazquez et al., 2000) and 5-HT1A receptor binding densities was observed in the hippocampus of rat pups (Vazquez et al., 2002) following 24 h of MS. The findings of changes in 5-HT1A receptor densities in pups coupled with our results in adults suggest that such changes might persist throughout life. In contrast to these and our findings, no changes in either mRNA or densities for SERT and 5-HT1A receptors were observed in dorsal raphe nucleus and CA1 hippocampal area as a consequence of MS (Arborelius et al., 2004). However, these investigators examined only MS15 and MS180 without including MS0, NH or AFR groups. Another study that compared only MS15 with MS180 group also did not observe alterations in 5-HT1A receptor mRNA levels in the dorsal raphe nucleus and CA1 hippocampal area in maternally separated groups (Neumaier et al., 2002). While the lack of changes in the CA1 hippocampal area agrees with our results, we did find changes in the CA3 region. However, the findings from ours and studies by others may be difficult to compare due to differences in species, age and sex as well as the separation paradigms employed. In spite of these differences, the bulk of the evidence including ours favors a significant change in serotonergic systems due to early life MS and variation in handling. Our observed changes in 5-HT1A binding are complemented by changes in 5-HT1A receptor function as measured by 8-OH-DPAT-induced plasma CORT and ACTH (Critchley et al., 1994; Hoyer et al., 1994; Vicentic et al., 1998). Moreover, a 8-OH-DPAT-induced decrease in 5-HT dialysate levels was significantly attenuated in animals exposed to a 6 h MS and this phenomenon was attributed to functional 5-HT1A receptor desensitization (Gartside et al., 2003). The reduction in CORT and ACTH found here, while small in magnitude, paralleled the reduction in binding and was found only in the NH group where the binding changes were observed. This indicates that the binding changes likely reflect functional changes as well. Another

possibility is that changes in the oxytocin system underlie changes in 5-HT1A receptor densities. Previous findings demonstrated that a low level of maternal licking/grooming, lack of arched-back nursing and aggression correlated with the MS phenotype and was inheritable to female offspring (Boccia and Pedersen, 2001; Champagne and Meaney, 2001; Bosch et al., 2005). Because oxytocin stimulates positive maternal behaviors and diminishes anxiety, aggression and stress, they hypothesized that change in central oxytocin receptors in mothers may link the MS phenotype to the behaviors observed in the offspring. The measurements of maternal behaviors or female offspring were not within the scope of this study. However, based on this evidence and the role of 5-HT1A receptors in oxytocin secretion (Jorgensen et al., 2003; Osei-Owusu et al., 2005), changes in oxytocin receptors or release in male offspring of the NH and MS180 mothers might have accounted for the observed changes in 5-HT1A receptors. Observed differences in SERT and 5-HT1A receptor densities and function are relevant in that our previous study of alcohol intake utilized the same five groups for comparison (Jaworski et al., 2005). This study demonstrated that MS15 exhibits lower ethanol preference compared with the NH and MS180 while the NH group exhibited highest ethanol preference and greatest reduction in GABA receptor densities. In light of these results, it is worth noting that a short allelic variants of SERT appear to contribute to alcohol dependence (Hallikainen et al., 1999; Feinn et al., 2005), and that SERT densities are attenuated in adult alcoholics (Heinz et al., 1998) and in children that were prenatally exposed to alcohol (Riikonen et al., 2005). Thus, our results further support the hypothesis that changes in SERT densities are an important factor in alcohol dependence. Moreover, because of the role of SERT, 5-HT1A and other receptors in the action of cocaine (Walsh and Cunningham, 1997; Bubar et al., 2004; Andrews et al., 2005; Czoty et al., 2005), we expect to see differences in cocaine self-administration among the groups as well; our preliminary data indicate that this is so (not shown). Our findings that both SERT and 5-HT1A densities change raise the question of a possible link between the two, and such a link is clearly possible. There is a functional connection in that SERT obviously regulates 5-HT levels in the synapse and therefore indirectly regulates the activity of 5-HT1A autoreceptors. Indeed, continued administration of SSRIs is postulated to induce an early activation of 5-HT1A autoreceptors that later leads to a downregulation of these receptor densities (Blier and de Montigny, 1987; Chaput et al., 1991; Burnet et al., 1994). This may be analogous to our experiments where prolonged reduction of SERT densities and/or activity which occurs in the NH group could lead to down-regulation of 5-HT1A receptors by similar mechanisms. In the MS15 group where there is an up-regulation of SERT in the amygdala, there is a corresponding up-regulation of 5-HT1A receptors in the same region. While this does not prove a cause– effect relationship for SERT and 5-HT1A receptor levels in

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our animals, there is precedent for it and it is therefore a reasonable hypothesis. These animals may be of value in exploring antidepressant mechanisms or may be models of psychiatric disease. SERT and 5-HT1A levels are decreased in depressed humans for example (Malison et al., 1998; Hsiung et al., 2003; Bhagwagar et al., 2004). Indeed, SERT gene promoter variations rendered blunted CORT response in rhesus monkeys exposed to perinatal stress (Barr et al., 2004) while transient inhibition of SERT during early development with SSRIs increased anxiety levels in adult animals (Ansorge et al., 2004). SERT and/or 5-HT1A receptor binding and function are decreased in a variety of human psychiatric disorders (Nemeroff et al., 1994; Marazziti et al., 1995; Coccaro et al., 1996; Malison et al., 1998; Hsiung et al., 2003; Bhagwagar et al., 2004) including alcoholism (Heinz et al., 1998; Riikonen et al., 2005). Moreover, because SERT and 5-HT1A play such an important role in the mechanisms of action of SSRIs, perinatal treatment-induced changes in their densities and function are relevant to possible alterations in therapeutic efficacy of SSRIs in individuals exposed to early life adversities.

CONCLUSION In summary, we show that not only time of separation but also the degree of handling during first two weeks of life could affect both the densities and function of 5-HT1A receptors and the densities of SERT in many limbic forebrain regions. Additional studies with this model may help clarify how early rearing conditions might affect the serotonergic system and thus potentially increase vulnerability for behavioral disorders in later life. Future experiments will examine whether some pharmacological treatments could reverse some of the observed changes in SERT and 5-HT1A receptors.

REFERENCES Anand KJ, Scalzo FM (2000) Can adverse neonatal experiences alter brain development and subsequent behavior? Biol Neonate 77:69 – 82. Andrews CM, Kung HF, Lucki I (2005) The 5-HT1A receptor modulates the effects of cocaine on extracellular serotonin and dopamine levels in the nucleus accumbens. Eur J Pharmacol 508: 123–130. Ansorge MS, Zhou M, Lira A, Hen R, Gingrich JA (2004) Early-life blockade of the 5-HT transporter alters emotional behavior in adult mice. Science 306:879 – 881. Arborelius L, Hawks BW, Owens MJ, Plotsky PM, Nemeroff CB (2004) Increased responsiveness of presumed 5-HT cells to citalopram in adult rats subjected to prolonged maternal separation relative to brief separation. Psychopharmacology (Berl) 176:248 –255. Arborelius L, Nomikos GG, Hertel P, Salmi P, Grillner P, Hook BB, Hacksell U, Svensson TH (1996) The 5-HT1A receptor antagonist (S)-UH-301 augments the increase in extracellular concentrations of 5-HT in the frontal cortex produced by both acute and chronic treatment with citalopram. Naunyn Schmiedebergs Arch Pharmacol 353:630 – 640. Arborelius L, Owens MJ, Plotsky PM, Nemeroff CB (1999) The role of corticotropin-releasing factor in depression and anxiety disorders. J Endocrinol 160:1–12.

363

Baldwin D, Rudge S (1995) The role of serotonin in depression and anxiety. Int Clin Psychopharmacol 9 (Suppl 4):41– 45. Barr CS, Newman TK, Shannon C, Parker C, Dvoskin RL, Becker ML, Schwandt M, Champoux M, Lesch KP, Goldman D, Suomi SJ, Higley JD (2004) Rearing condition and rh5-HTTLPR interact to influence limbic-hypothalamic-pituitary-adrenal axis response to stress in infant macaques. Biol Psychiatry 55:733–738. Bhagwagar Z, Rabiner EA, Sargent PA, Grasby PM, Cowen PJ (2004) Persistent reduction in brain serotonin1A receptor binding in recovered depressed men measured by positron emission tomography with [11C]WAY-100635. Mol Psychiatry 9:386 –392. Blier P, de Montigny C (1987) Modification of 5-HT neuron properties by sustained administration of the 5-HT1A agonist gepirone: electrophysiological studies in the rat brain. Synapse 1:470 – 480. Boccia ML, Pedersen CA (2001) Brief vs. long maternal separations in infancy: contrasting relationships with adult maternal behavior and lactation levels of aggression and anxiety. Psychoneuroendocrinology 26:657– 672. Boja JW, Mitchell WM, Patel A, Kopajtic TA, Carroll FI, Lewin AH, Abraham P, Kuhar MJ (1992) High-affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain. Synapse 12: 27–36. Bosch OJ, Meddle SL, Beiderbeck DI, Douglas AJ, Neumann ID (2005) Brain oxytocin correlates with maternal aggression: link to anxiety. J Neurosci 25:6807– 6815. Brake WG, Zhang TY, Diorio J, Meaney MJ, Gratton A (2004) Influence of early postnatal rearing conditions on mesocorticolimbic dopamine and behavioural responses to psychostimulants and stressors in adult rats. Eur J Neurosci 19:1863–1874. Bremne JD, Vermetten E (2001) Stress and development: behavioral and biological consequences. Dev Psychopathol 13:473– 489. Bubar MJ, Pack KM, Frankel PS, Cunningham KA (2004) Effects of dopamine D1- or D2-like receptor antagonists on the hypermotive and discriminative stimulus effects of (⫹)-MDMA. Psychopharmacology (Berl) 173:326 –336. Burnet PW, Michelson D, Smith MA, Gold PW, Sternberg EM (1994) The effect of chronic imipramine administration on the densities of 5-HT1A and 5-HT2 receptors and the abundances of 5-HT receptor and transporter mRNA in the cortex, hippocampus and dorsal raphe of three strains of rat. Brain Res 638:311–324. Caldji C, Diorio J, Meaney MJ (2000a) Variations in maternal care in infancy regulate the development of stress reactivity. Biol Psychiatry 48:1164 –1174. Caldji C, Francis D, Sharma S, Plotsky PM, Meaney MJ (2000b) The effects of early rearing environment on the development of GABAA and central benzodiazepine receptor levels and novelty-induced fearfulness in the rat. Neuropsychopharmacology 22:219 –229. Celada P, Puig M, Amargos-Bosch M, Adell A, Artigas F (2004) The therapeutic role of 5-HT1A and 5-HT2A receptors in depression. J Psychiatry Neurosci 29:252–265. Champagne F, Meaney MJ (2001) Like mother, like daughter: evidence for non-genomic transmission of parental behavior and stress responsivity. Prog Brain Res 133:287–302. Champagne FA, Weaver IC, Diorio J, Sharma S, Meaney MJ (2003) Natural variations in maternal care are associated with estrogen receptor alpha expression and estrogen sensitivity in the medial preoptic area. Endocrinology 144:4720 – 4724. Chaput Y, de Montigny C, Blier P (1991) Presynaptic and postsynaptic modifications of the serotonin system by long-term administration of antidepressant treatments. An in vivo electrophysiologic study in the rat. Neuropsychopharmacology 5:219 –229. Coccaro EF, Kavoussi RJ, Sheline YI, Lish JD, Csernansky JG (1996) Impulsive aggression in personality disorder correlates with tritiated paroxetine binding in the platelet. Arch Gen Psychiatry 53: 531–536. Critchley DJ, Childs KJ, Middlefell VC, Dourish CT (1994) Inhibition of 8-OH-DPAT-induced elevation of plasma corticotrophin by the

364

A. Vicentic et al. / Neuroscience 140 (2006) 355–365

5-HT1A receptor antagonist WAY100635. Eur J Pharmacol 264: 95–97. Czoty PW, McCabe C, Nader MA (2005) Effects of the 5-HT(1A) agonist (⫹/⫺)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) on cocaine choice in cynomolgus monkeys. Behav Pharmacol 16:187–191. Daniels WM, Pietersen CY, Carstens ME, Stein DJ (2004) Maternal separation in rats leads to anxiety-like behavior and a blunted ACTH response and altered neurotransmitter levels in response to a subsequent stressor. Metab Brain Dis 19:3–14. Dawson LA, Nguyen HQ, Smith DL, Schechter LE (2002) Effect of chronic fluoxetine and WAY-100635 treatment on serotonergic neurotransmission in the frontal cortex. J Psychopharmacol 16: 145–152. Denenberg VH (1999) Commentary: is maternal stimulation the mediator of the handling effect in infancy? Dev Psychobiol 34:1–3. D’Souza DN, Zhang Y, Garcia F, Battaglia G, Van de Kar LD (2004) Fluoxetine-induced changes in body weight and 5-HT1A receptormediated hormone secretion in rats on a tryptophan-deficient diet. Am J Physiol Regul Integr Comp Physiol 286:R390 –R397. Feinn R, Nellissery M, Kranzler HR (2005) Meta-analysis of the association of a functional serotonin transporter promoter polymorphism with alcohol dependence. Am J Med Genet B Neuropsychiatr Genet 133:79 – 84. Francis D, Diorio J, Liu D, Meaney MJ (1999) Nongenomic transmission across generations of maternal behavior and stress responses in the rat. Science 286:1155–1158. Francis DD, Diorio J, Plotsky PM, Meaney MJ (2002) Environmental enrichment reverses the effects of maternal separation on stress reactivity. J Neurosci 22:7840 –7843. Gartside SE, Johnson DA, Leitch MM, Troakes C, Ingram CD (2003) Early life adversity programs changes in central 5-HT neuronal function in adulthood. Eur J Neurosci 17:2401–2408. Greisen MH, Bolwig TG, Husum H, Nedergaard P, Wortwein G (2005) Maternal separation affects male rat copulatory behaviour and hypothalamic corticotropin releasing factor in concert. Behav Brain Res 158:367–375. Hall FS (1998) Social deprivation of neonatal, adolescent, and adult rats has distinct neurochemical and behavioral consequences. Crit Rev Neurobiol 12:129 –162. Hallikainen T, Saito T, Lachman HM, Volavka J, Pohjalainen T, Ryynanen OP, Kauhanen J, Syvalahti E, Hietala J, Tiihonen J (1999) Association between low activity serotonin transporter promoter genotype and early onset alcoholism with habitual impulsive violent behavior. Mol Psychiatry 4:385–388. Heils A, Mossner R, Lesch KP (1997) The human serotonin transporter gene polymorphism-basic research and clinical implications. J Neural Transm 104:1005–1014. Heim C, Nemeroff CB (2001) The role of childhood trauma in the neurobiology of mood and anxiety disorders: preclinical and clinical studies. Biol Psychiatry 49:1023–1039. Heim C, Newport DJ, Bonsall R, Miller AH, Nemeroff CB (2001) Altered pituitary-adrenal axis responses to provocative challenge tests in adult survivors of childhood abuse. Am J Psychiatry 158: 575–581. Heinz A, Jones DW, Mazzanti C, Goldman D, Ragan P, Hommer D, Linnoila M, Weinberger DR (2000) A relationship between serotonin transporter genotype and in vivo protein expression and alcohol neurotoxicity. Biol Psychiatry 47:643– 649. Heinz A, Ragan P, Jones DW, Hommer D, Williams W, Knable MB, Gorey JG, Doty L, Geyer C, Lee KS, Coppola R, Weinberger DR, Linnoila M (1998) Reduced central serotonin transporters in alcoholism. Am J Psychiatry 155:1544 –1549. Hensler JG (2002) Differential regulation of 5-HT1A receptor-G protein interactions in brain following chronic antidepressant administration. Neuropsychopharmacology 26:565–573. Higley JD, Suomi SJ, Linnoila M (1996) A nonhuman primate model of type II alcoholism? Part 2. Diminished social competence and excessive aggression correlates with low cerebrospinal fluid 5-hy-

droxyindoleacetic acid concentrations. Alcohol Clin Exp Res 20: 643– 650. Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, Saxena PR, Humphrey PP (1994) International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin). Pharmacol Rev 46:157–203. Hsiung SC, Adlersberg M, Arango V, Mann JJ, Tamir H, Liu KP (2003) Attenuated 5-HT1A receptor signaling in brains of suicide victims: involvement of adenylyl cyclase, phosphatidylinositol 3-kinase, Akt and mitogen-activated protein kinase. J Neurochem 87:182–194. Huot RL, Plotsky PM, Lenox RH, McNamara RK (2002) Neonatal maternal separation reduces hippocampal mossy fiber density in adult Long Evans rats. Brain Res 950:52– 63. Huot RL, Thrivikraman KV, Meaney MJ, Plotsky PM (2001) Development of adult ethanol preference and anxiety as a consequence of neonatal maternal separation in Long Evans rats and reversal with antidepressant treatment. Psychopharmacology (Berl) 158:366 – 373. Jaworski JN, Francis DD, Brommer CL, Morgan ET, Kuhar MJ (2005) Effects of early maternal separation on ethanol intake, GABA receptors and metabolizing enzymes in adult rats. Psychopharmacology (Berl) 181:8 –15. Jorgensen H, Kjaer A, Knigge U, Moller M, Warberg J, Riis M (2003) Serotonin stimulates hypothalamic mRNA expression and local release of neurohypophysial peptides: serotonin receptors involved in vasopressin and oxytocin secretion. J Neuroendocrinol 15:564 –571. Kalinichev M, Easterling KW, Holtzman SG (2003) Long-lasting changes in morphine-induced locomotor sensitization and tolerance in Long-Evans mother rats as a result of periodic postpartum separation from the litter: a novel model of increased vulnerability to drug abuse? Neuropsychopharmacology 28:317–328. Kasper S, Tauscher J, Willeit M, Stamenkovic M, Neumeister A, Kufferle B, Barnas C, Stastny J, Praschak-Rieder N, Pezawas L, de Zwaan M, Quiner S, Pirker W, Asenbaum S, Podreka I, Brucke T (2002) Receptor and transporter imaging studies in schizophrenia, depression, bulimia and Tourette’s disorder-implications for psychopharmacology. World J Biol Psychiatry 3:133–146. Kinney GG, Taber MT, Gribkoff VK (2000) The augmentation hypothesis for improvement of antidepressant therapy: is pindolol a suitable candidate for testing the ability of 5HT1A receptor antagonists to enhance SSRI efficacy and onset latency? Mol Neurobiol 21:137–152. Kung MP, Frederick D, Mu M, Zhuang ZP, Kung HF (1995) 4-(2=Methoxy-phenyl)-1-[2=-(n-2=-pyridinyl)-p-iodobenzamido]-ethyl-piperazine ([125I]p-MPPI) as a new selective radioligand of serotonin-1A sites in rat brain: in vitro binding and autoradiographic studies. J Pharmacol Exp Ther 272:429 – 437. Li Q, Holmes A, Ma L, Van de Kar LD, Garcia F, Murphy DL (2004) Medial hypothalamic 5-hydroxytryptamine (5-HT)1A receptors regulate neuroendocrine responses to stress and exploratory locomotor activity: application of recombinant adenovirus containing 5-HT1A sequences. J Neurosci 24:10868 –10877. Li Q, Wichems C, Heils A, Lesch KP, Murphy DL (2000) Reduction in the density and expression, but not G-protein coupling, of serotonin receptors (5-HT1A) in 5-HT transporter knock-out mice: gender and brain region differences. J Neurosci 20:7888 –7895. Li Q, Wichems C, Heils A, Van De Kar LD, Lesch KP, Murphy DL (1999) Reduction of 5-hydroxytryptamine (5-HT)(1A)-mediated temperature and neuroendocrine responses and 5-HT(1A) binding sites in 5-HT transporter knockout mice. J Pharmacol Exp Ther 291:999 –1007. 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. Madruga C, Xavier LL, Achaval M, Sanvitto GL, Lucion AB (2005) Early handling, but not maternal separation, decreases emotional

A. Vicentic et al. / Neuroscience 140 (2006) 355–365 responses in two paradigms of fear without changes in mesolimbic dopamine. Behav Brain Res 166:241–246. Maes M, Vandoolaeghe E, Desnyder R (1996) Efficacy of treatment with trazodone in combination with pindolol or fluoxetine in major depression. J Affect Disord 41:201–210. Malison RT, Price LH, Berman R, van Dyck CH, Pelton GH, Carpenter L, Sanacora G, Owens MJ, Nemeroff CB, Rajeevan N, Baldwin RM, Seibyl JP, Innis RB, Charney DS (1998) Reduced brain serotonin transporter availability in major depression as measured by [123I]-2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane and single photon emission computed tomography. Biol Psychiatry 44: 1090 –1098. Marazziti D, Palego L, Dal Canto B, Rotondo A, Pasqualetti M, Gino G, Lucacchini A, Ladinsky H, Nardi I, Cassano GB (1995) Presence of serotonin1A (5-HT1A) receptor mRNA without binding of [3H]-8OH-DPAT in peripheral blood mononuclear cells. Life Sci 57: 2197–2203. McGregor IS, Clemens KJ, Van der Plasse G, Li KM, Hunt GE, Chen F, Lawrence AJ (2003) Increased anxiety 3 months after brief exposure to MDMA (“ecstasy”) in rats: association with altered 5-HT transporter and receptor density. Neuropsychopharmacology 28:1472–1484. Meaney MJ (2001) Maternal care, gene expression, and the transmission of individual differences in stress reactivity across generations. Annu Rev Neurosci 24:1161–1192. Meaney MJ, Brake W, Gratton A (2002) Environmental regulation of the development of mesolimbic dopamine systems: a neurobiological mechanism for vulnerability to drug abuse? Psychoneuroendocrinology 27:127–138. Mellerup E, Bennike B, Bolwig T, Dam H, Hasholt L, Jorgensen MB, Plenge P, Sorensen SA (2001) Platelet serotonin transporters and the transporter gene in control subjects, unipolar patients and bipolar patients. Acta Psychiatr Scand 103:229 –233. Nemeroff CB, Knight DL, Franks J, Craighead WE, Krishnan KR (1994) Further studies on platelet serotonin transporter binding in depression. Am J Psychiatry 151:1623–1625. Neumaier JF, Edwards E, Plotsky PM (2002) 5-HT(1B) mRNA regulation in two animal models of altered stress reactivity. Biol Psychiatry 51:902–908. Osei-Owusu P, James AE, Crane J, Scrogin KE (2005) 5-HT1A receptors in the paraventricular nucleus of the hypothalamus mediate oxytocin and adrenocorticotropin hormone release as well as some behavioral components of the serotonin syndrome. J Pharmacol Exp Ther 313:1324 –1330. Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates. San Diego, CA: Academic Press, Inc. Ploj K, Roman E, Nylander I (2003) Long-term effects of maternal separation on ethanol intake and brain opioid and dopamine receptors in male Wistar rats. Neuroscience 121:787–799. Riikonen RS, Nokelainen P, Valkonen K, Kolehmainen AI, Kumpulainen KI, Kononen M, Vanninen RL, Kuikka JT (2005) Deep serotonergic and dopaminergic structures in fetal alcoholic syndrome: a study with nor-beta-CIT-single-photon emission com-

365

puted tomography and magnetic resonance imaging volumetry. Biol Psychiatry 57:1565–1572. Ruedi-Bettschen D, Pedersen EM, Feldon J, Pryce CR (2005) Early deprivation under specific conditions leads to reduced interest in reward in adulthood in Wistar rats. Behav Brain Res 156:297–310. Sacristan JA, Gilaberte I, Boto B, Buesching DP, Obenchain RL, Demitrack M, Perez Sola V, Alvarez E, Artigas F (2000) Costeffectiveness of fluoxetine plus pindolol in patients with major depressive disorder: results from a randomized, double-blind clinical trial. Int Clin Psychopharmacol 15:107–113. Sadowski H, Ugarte B, Kolvin I, Kaplan C, Barnes J (1999) Early life family disadvantages and major depression in adulthood. Br J Psychiatry 174:112–120. Stoltenberg SF, Twitchell GR, Hanna GL, Cook EH, Fitzgerald HE, Zucker RA, Little KY (2002) Serotonin transporter promoter polymorphism, peripheral indexes of serotonin function, and personality measures in families with alcoholism. Am J Med Genet 114: 230 –234. Tjurmina OA, Armando I, Saavedra JM, Goldstein DS, Murphy DL (2002) Exaggerated adrenomedullary response to immobilization in mice with targeted disruption of the serotonin transporter gene. Endocrinology 143:4520 – 4526. Vazquez DM, Eskandari R, Zimmer CA, Levine S, Lopez JF (2002) Brain 5-HT receptor system in the stressed infant rat: implications for vulnerability to substance abuse. Psychoneuroendocrinology 27:245–272. 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. Vicentic A, Li Q, Battaglia G, Van de Kar LD (1998) WAY-100635 inhibits 8-OH-DPAT-stimulated oxytocin, ACTH and corticosterone, but not prolactin secretion. Eur J Pharmacol 346:261– 266. Walsh SL, Cunningham KA (1997) Serotonergic mechanisms involved in the discriminative stimulus, reinforcing and subjective effects of cocaine [review]. Psychopharmacology 130:41–58. Zaharia MD, Kulczycki J, Shanks N, Meaney MJ, Anisman H (1996) The effects of early postnatal stimulation on Morris water-maze acquisition in adult mice: genetic and maternal factors. Psychopharmacology (Berl) 128:227–239. Zalsman G, Anderson GM, Peskin M, Frisch A, King RA, Vekslerchik M, Sommerfeld E, Michaelovsky E, Sher L, Weizman A, Apter A (2005) Relationships between serotonin transporter promoter polymorphism, platelet serotonin transporter binding and clinical phenotype in suicidal and non-suicidal adolescent inpatients. J Neural Transm 112:309 –315. Ziabreva I, Poeggel G, Schnabel R, Braun K (2003a) Separationinduced receptor changes in the hippocampus and amygdala of Octodon degus: influence of maternal vocalizations. J Neurosci 23:5329 –5336. Ziabreva I, Schnabel R, Poeggel G, Braun K (2003b) Mother’s voice “buffers” separation-induced receptor changes in the prefrontal cortex of Octodon degus. Neuroscience 119:433– 441.

(Accepted 4 February 2006) (Available online 13 March 2006)