HIPPOCAMPUS 24:493–501 (2014)

Subregion-Specific Decreases in Hippocampal Serotonin Transporter Protein Expression and Function Associated With Endophenotypes of Depression Man Tang,1* Tao He,1 Xiao Sun,2 Qing-Yan Meng,3 Yao Diao,4 Jie-Yu Lei,1 Xiao-Jing He,5 Lei Chen,6 Xiu-Bo Sang,7 and Shulei Zhao8

ABSTRACT: Stress influences the development of depression, and depression is associated with structural and functional changes in the hippocampus. The current study sought to determine whether chronic corticosteroid (CORT) treatment influences serotonin transporter (5-HTT) protein expression and function in the CA1, CA3, and dentate gyrus (DG) subregions of the hippocampus. Male CD-1 mice were subcutaneously injected with CORT at a dose of 20 mg/kg once daily for 3 weeks. Behavioral state was assessed using sucrose preference, physical state of the coat, forced swimming test, and tail suspension test. We then determine 5-HTT protein expression and synaptosomal 5-HT uptake in the CA1, CA3 and DG subregions. CORT treatment induced anhedonia and behavioral despair, two core endophenotypes of clinical depression; 5-HTT protein expression levels and synaptosomal 5-HT uptake were both decreased in a subregion-specific manner, with the greatest decrease observed in the DG, a moderate decrease in the CA3, and the CA1 showed no apparent change. In addition, a reduction in tissue mass was detected in the DG following the CORT treatment. These data indicate that subregion-specific decreases in hippocampal 5-HTT protein expression and function are associated with endophenoC 2014 Wiley Periodicals, Inc. types of depression. V KEY WORDS: anhedonia; behavioral despair; corticosteroid; hippocampus; synaptosomal 5-HT uptake

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Department of Clinical Pharmacology, School of Pharmacy, China Medical University, Shenyang, China; 2 Department of Internal Medicine, Shenyang Medical College Affiliated Fengtian Hospital, Shenyang, China; 3 Department of Burns, The Northern Hospital, Shenyang, China; 4 Center of Experiment and Technology, China Medical University, Shenyang, China; 5 Department of Pharmacy, Shengjing Hospital of China Medical University, Shenyang, China; 6 Department of Pharmacology, School of Pharmacy, China Medical University, Shenyang, China; 7 Department of Seven-year Clinical Medicine, China Medical University, Shenyang, China; 8 Section of Integrative Biology, School of Biological Sciences, University of Texas, Austin, Texas Grant sponsor: Liaoning Provincial Natural Science Foundation of China; Grant numbers: 201102276 and 20092073; Grant sponsor: Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry; Grant number: [2012]940; Grant sponsor: Provincial Innovative and Entrepreneurship Training Program for College Students (2013) (to XB Sang). *Correspondence to: Man Tang, Department of Clinical Pharmacology, School of Pharmacy, China Medical University, No. 92 Beier Road, Heping District, 110001, Shenyang, China. E-mail: [email protected] Accepted for publication 9 January 2014. DOI 10.1002/hipo.22242 Published online 16 January 2014 in Wiley Online Library (wileyonlinelibrary.com). C 2014 WILEY PERIODICALS, INC. V

INTRODUCTION Depression is a severe psychiatric illness with a life prevalence of approximately 16%, affecting up to 20% of global population (Kessler et al., 2003; Berton and Nestler, 2006; Krishnan and Nestler, 2008). The disease is defined by a constellation of different classes of symptoms, suggesting that multiple neural substrates and mechanisms may contribute to its pathophysiology. Hippocampus, a key limbic structure, has been extensively studied in patients with depression. A significant decrease in hippocampal tissue volume has been found in patients with recurrent depression (Campbell et al., 2004; Videbech and Ravnkilde, 2004). The magnitude of the reduction in hippocampal tissue volume correlates with the frequency of depressive episodes and the duration of untreated depression (MacQueen et al., 2003). Importantly, dysregulation of the hypothalamicpituitary-adrenal (HPA) axis and memory deficits, which are normally regulated by hippocampus, have been reported in depressive patients (Carroll et al., 1968; Sapolsky, 2000; Gould et al., 2007). The hippocampus receives convergent input from multiple afferent pathways, including the serotonergic (5-HT) neurons in the raphe nucleus. The serotonin transporter (5-HTT), located mainly on the 5-HT axonal terminal membrane, regulates the extracellular concentration of 5-HT to influence neurotransmission within the hippocampus. Recent studies show that a functional polymorphism in the human 5-HTT gene promoter (the 5-HTT gene-linked polymorphic region, 5-HTTLPR) is linked to an increased risk of developing major depression, linked to poor memory function, and linked to reduced hippocampal tissue volume (Caspi et al., 2003; O’Hara et al., 2007; Daniele et al., 2011). Moreover, neuroimaging studies show that hippocampal 5-HTT binding is altered in patients with depression (Newberg et al., 2012 ; Savitz and Drevets, 2012). Our previous work showed a down-regulation of hippocampal 5-HTT protein expression in mice that exhibited anhedonia, a core endophenotype of clinical depression. The anhedonia was induced by unpredictable chronic mild stress (UCMS) and was reversed by fluoxetine (FLX) treatment (Tang et al., 2013). Stress-

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induced changes in 5-HTT protein expression suggest the involvement of corticosteroid (CORT) stress pathways. The current study sought to determine whether chronic CORT treatment similarly influenced the protein expression of hippocampal 5-HTT. Clinical and animal studies reported little information on the functional changes of 5-HTT in depressive state. Hence, the second objective of the present study was to measure 5-HT uptake. In addition, the main subregions of the hippocampus (the CA1, CA3, and DG) are differentially involved in the regulation of emotional response, learning and memory, and the HPA axis (Swanson and Cowan, 1977; Herman et al., 1995; Moser and Moser, 1998; Pitkanen et al., 2000). Most recently, subregion-specific neurogenesis, i.e., neurogenesis in the DG, has been proposed to mediate anti-depressant action (Santarelli et al., 2003; Jiang et al., 2005; Airan et al., 2007). In view of these findings, we hypothesize that both protein expression and function of hippocampal 5-HTT are decreased in a subregionspecific manner in the depressive state. To test this hypothesis, we treated mice with CORT for 3 weeks to induce a depressive state (Zhao et al., 2008), then dissected the hippocampal CA1, CA3, and DG subregions to measure 5-HTT protein expression and synaptosomal 5-HT uptake.

MATERIALS AND METHODS Animals Male CD-1 mice (10–12 weeks of age) were used. Two weeks before experiments, each mouse was housed individually in a temperature (21 6 1 C) and humidity (55 6 2%) controlled room with a 12 h light-dark cycle (lights on at 8 A.M.). Animals were given free access to food and water for the duration of the experiments unless otherwise specified. All experimental procedures were carried out in accordance with the guidelines for humane care of animals set forth by the National Institute of Health (NIH) in the United States and approved by the Institutional Animal Care and Use Committee at China Medical University.

Experimental Design Thirty-six mice were randomly assigned to three groups (12/ group). One was served as the baseline group to measure the 5-HTT protein expression (n 5 6) and function (n 5 6) before the CORT treatment. The other two groups were given either CORT or vehicle for continuous 3 weeks. CORT was suspended in vehicle (saline containing 0.1% dimethylsulfoxide (DMSO) and 0.1% Tween-80) and given to the animals through subcutaneous injection at a dose of 20 mg/kg per day (between 8 and 10 A.M.) in a volume of 5 ml/kg (Zhao et al., 2008). Sucrose preference test, physical state of the coat, forced swimming test (FST), and tail suspension test (TST) were perHippocampus

formed or scored weekly. Each mouse in both the CORT and vehicle treatment groups was tested three times in each of the behavioral tests over the course of each treatment. Tissue mass, 5-HTT protein expression, Vm and Km derived from synaptosomal [3H]5-HT uptake assay of each hippocampal subregion were measured at the baseline and following the 3 weeks of CORT treatment.

Sucrose Preference Test Mice were given two standard drinking bottles containing 2.5% sucrose or tap water. The night before the sucrose preference test, mice were food and water deprived. The two bottles were presented next morning between 8:00 and 9:00 A.M., with the positions of the two bottles (right/left) interchanged randomly from trial to trial to eliminate possible effects of side-preference on drinking behavior. Intake of each solution was determined by the difference in bottle weight measured between pre- and post-drinking session. Sucrose preference (%) was calculated as: [sucrose solution intake (ml)/total fluid intake (ml)] 3 100.

Physical State of the Coat Grooming behavior was assessed by monitoring the physical state of the coat. The coat in six areas of the body (head, neck, back, belly, forepaws, hindpaws) was scored as previously described (Stemmelin et al., 2010): 0 for normal physical state; 0.5 for medium degradation; 1 for high degradation. An overall measurement of the coat state was obtained by summation of the scores from the six different areas.

Forced Swimming Test The test was performed with a few modifications based on the procedure described by (Porsolt et al., 1977). Mice were placed individually for 6 mins into plastic cylinder (height 5 45 cm, diameter 5 19 cm) containing water (height 5 23 cm), maintained at 23 6 1 C, and were scored for immobility during the last 4 mins. Immobility was defined as the absence of active, escape-oriented behaviors, only with small movements to keep its head above water.

Tail Suspension Test The procedure was similar to that described previously (Steru et al., 1985). Mice were individually suspended from the edge of a shelf 60 cm above the ground by adhesive tape placed over 1 cm from the tip of the tail. Mice were hung for 6 mins, and were scored for immobility during the last 4 mins. Immobility was defined as absence of motion without any agitation.

Dissection of Hippocampal Subregions Mice were decapitated. Hippocampi were quickly removed on ice, and further dissected along the visible boundaries of DG to yield CA1, CA3, and DG (Lein et al., 2004).

SUBREGION-SPECIFIC DECREASES IN HIPPOCAMPAL 5-HTT IN DEPRESSIVE STATE

Tissue Mass For each mouse, the three dissected hippocampal subregions were weighted and recorded, then subject to Western blot of 5-HTT or synaptosomal [3H]5-HT uptake assay, respectively.

Western Blot of 5-HTT For each mouse, the dissected hippocampal subregion was homogenized in ice-cold lysis buffer (0.1% SDS, 1% Triton100, 10 mM Hepes, 5 mM NaF, 0.25 M sucrose, pH 7.4) to make cell lysates. Protein concentration was measured using the Bradford method (Bradford, 1976), with bovine albumin as the standard. Samples containing 100 lg of protein were loaded onto 12% polyacrylamide gel, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) overnight. The membrane was blocked with 5% milk in Tris-buffered saline with Tween-20 (TBS-T) for 2 h at room temperature. A goat anti-5-HTT polyclonal antibody (1:300, Santa Cruz) was incubated with the membrane for four hours at room temperature. The membrane was washed once for 30 min and twice for 20 min with TBS-T, and incubated with a horseradish peroxidase-conjugated anti-goat IgG secondary antibody (1:500, Santa Cruz) for 2 h at room temperature. After the same washing steps, the specific immunoreactive staining was visualized by enhanced chemiluminescence (ECL) detection reagents (Sigma), and captured by Tanon—4200 Gel Imaging System (Tanon Science, China). b-actin, a housekeeping protein, was detected with a rabbit anti-b-actin polyclonal antibody (1:1,000, Santa Cruz) and secondary anti-rabbit antibody (1:1,000, Santa Cruz). 5-HTT was revealed as a band of 70 KDa, b-actin as a band of 42 KDa. Band density was analyzed with GIS image analysis software (Tanon Science, China). To eliminate possible variations in the efficiency of protein extractions and sample loading, b-actin was used as an internal control. Hence, the expression level of 5-HTT was normalized to its corresponding b-actin level in each sample.

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Each synaptosomal suspension was stored at 4 C, then used for the 5-HT uptake assay within one hour. Protein concentration was measured using the Bradford method (Bradford, 1976). The uptake of 5-HT was measured using five different concentrations of [3H]5-HT (100 Ci/mmol, Perkin Elmer): 20, 40, 60, 80, 100 nM. Tubes containing different concentrations of [3H]5-HT were pre-incubated in 470 ll of the incubation buffer for five minutes at 37 C. Thirty microliters of the synaptosomal suspension were added to each tube and incubated for 10 min at 37 C with gentle agitation. The reaction was terminated by immersing the tubes in ice water followed by rapid filtration through Whatman GF/B filters. The filters were washed with incubation buffer and the radioactivity trapped on the filters was counted using a Packard 1900 TR liquid scintillation analyzer (Biostad). Specific [3H]5-HT uptake was defined as the difference between the total uptake at 37 C and the uptake in the presence of 0.1 mM fluoxetine at 0 C, and expressed as pmol/ mg protein/min. The data were fit to a Michaelis-Menten equation to determine the parameters of 5-HT saturation kinetics: Vmax and Km, which are the maximal uptake capacity and uptake efficiency of the membrane-bound 5-HTT, respectively.

Statistical Analysis Sucrose preference, physical state of the coat, and immobility time were analyzed using random block ANOVA with one between-subject variable (treatment) and one within-subject variable (week). Simple effect tests with Bonferroni correction factors were performed to distinguish the influence of one variable on another when a significant interaction was detected. For each hippocampal subregion, tissue mass, protein expression of 5-HTT, Vmax and Km were analyzed between the CORT- and vehicle-treated groups at the baseline and following the three-week treatment using one-way ANOVA. The level of significance was P < 0.05. Values are expressed as mean 6 SEM.

Synaptosomal [3H]5-HT Uptake For each mouse, the dissected hippocampal subregion was transferred into 15 volumes of ice-cold 0.32 M sucrose (pH 7.4, buffered with 5 mM Hepes), then homogenized at 800 r/min for 30 sec. In order to maximize the yield of milligram synaptic protein/ml from each hippocampal subregion and to preserve functional viability of synaptic 5-HTT, following procedures were performed. The tissue homogenates were first centrifuged at 1,000g (10 min, 4 C). The supernatants were then centrifuged at 17,000g (20 min, 4 C). The resulting pellets were resuspended in different volumes of incubation buffer (0.17 ml for the CA1, 0.18 ml for the CA3, and 0.16 ml for the DG) to a final protein concentration of 0.97 to 1.05 mg/ml. The incubation buffer contained 130 mM NaCl, 2.6 mM KCl, 1.2 mM MgSO4, 1.0 mM KH2PO4, 1.3 mM CaCl2, 25 mM NaHCO3, 10 mM glucose, 0.05 mM EDTA, 0.1 mM ascorbic acid, and 100 lM pargyline, at a pH of 7.4.

RESULTS CORT Treatment Induced Anhedonia as Measured by Sucrose Preference and Coat State Before the CORT treatment, the basal sucrose preferences were similar (81 6 4.7% for the CORT-treated group; 79 6 5.1% for the vehicle-treated group). Random block ANOVA on sucrose preference revealed a significant two-way interaction during the 3 weeks of treatment [treatment 3 week: F(3,66) 5 18.32, P < 0.05; n 5 12/group]. Simple effect tests showed that the 3 weeks of the CORT treatment induced a significant decrease in sucrose preference compared with their vehicletreated controls [treatment: F(1,22) 5 21.78, P < 0.05; week: F(3,66) 5 14.63, P < 0.05] (Fig. 1A). The scores indicating the physical state of the coat were similar between groups at the baseline (1.0 6 0.25 for the Hippocampus

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FIGURE 1. CORT treatment decreased sucrose preference and deteriorated the physical state of the coat. Repeated CORT injections induced a decrease in the sucrose preference (A), and a deterioration in the physical state of the coat (B). The vehicle treatment had no effect on either measurement (n 5 12/group; P > 0.05). *P < 0.05 versus corresponding vehicle-treated controls (n 5 12/group) by simple effects post hoc analysis after a significant two-way interaction in the overall ANOVA.

CORT-treated group; 0.98 6 0.24 for the vehicle-treated group). Random block ANOVA on the coat scores revealed a significant two-way interaction during the course of treatment [treatment x week: F(3,66) 5 9.32, P < 0.05; n 5 12/group]. Simple effect tests showed that two weeks into the CORT treatment, there was a significant deterioration in the physical state of the coat compared with the vehicle-treated controls [treatment: F(1,22) 5 11.78, P < 0.05; week: F(3,66) 5 9.63, P < 0.05] (Fig. 1B).

CORT Treatment Induced Behavioral Despair as Measured by FST and TST In the FST, immobility times for both groups of mice were similar at the baseline (42.0 6 10.1 sec for the CORT-treated group; 45.4 6 8.9 sec for the vehicle-treated group). During the CORT treatment, a significant two-way interaction was revealed by random block ANOVA [treatment 3 week: F(3,66) 5 17.42, P < 0.05; n 5 12/group], with significant increases in immobility time detected after two weeks of CORT treatment compared with the vehicle-treated controls [treatment: Hippocampus

FIGURE 2. CORT treatment increased the immobility time in both the forced swimming test and the tail suspension test. Repeated CORT injections increased the time spent immobile during the last 4 minutes of the FST (A), and the TST (B). The vehicle treatment has no effect on the immobility time in either test (n 5 12/group; P > 0.05). *P < 0.05 versus corresponding vehicletreated controls (n 5 12/group) by simple effects post hoc analysis after a significant two-way interaction or a significant treatment effect in the overall ANOVA.

F(1,22) 5 8.16, P < 0.05; week: F(3,66) 5 10.27, P < 0.05] (Fig. 2A). In the TST, basal immobility times were also similar (67.1 6 10.7 sec for the CORT-treated group; 64.0 6 8.7 sec for the vehicle-treated group). During the CORT treatment, no interaction was detected by random block ANOVA [treatment 3 week: F(3,66) 5 2.11, P > 0.05; n 5 12/group], and only a significant treatment effect was found [treatment: F(1,22) 5 12.67, P < 0.05]. Simple effect tests indicated that an increase in immobility time was only observed following 3 weeks of the CORT treatment (Fig. 2B).

CORT Treatment Decreased Tissue Mass in the DG At baseline, no differences in tissue mass were found between the CA1, CA3, and DG (F 5 1.08, P > 0.05; n 5 12). Prior to the treatment, tissue mass of each subregion was similar between the CORT-treated mice and the vehicle-treated controls (CA1: F 5 0.79, P > 0.05; CA3: F 5 0.86, P > 0.05; DG: F 5 0.55, P > 0.05, n 5 12/group). The tissue mass of each subregion was

SUBREGION-SPECIFIC DECREASES IN HIPPOCAMPAL 5-HTT IN DEPRESSIVE STATE

FIGURE 3. Tissue mass in the hippocampus following CORT treatment. Repeated CORT injections decreased the tissue mass in the DG, but not in the CA1 or CA3. The vehicle treatment did not alter tissue mass in any subregion of the hippocampus (n 5 6/group; P > 0.05). *P < 0.05 versus vehicle-treated controls by one-way ANOVA.

not changed after the vehicle treatment compared with baseline (CA1: F 5 0.67, P > 0.05; CA3: F 5 0.54, P > 0.05; DG: F 5 0.75, P > 0.05, n 5 12/group). In contrast, the CORT treatment caused a significant decrease in tissue mass in the DG (F 5 6.71, P < 0.05; n 5 12/group), but not in the CA1 or CA3 subregions (CA1: F 5 0.81, P > 0.05; CA3: F 5 0.67, P > 0.05; n 5 12/group), compared with their vehicle-treated controls (Fig. 3).

CORT Treatment Subregion-Specifically Decreased 5-HTT Protein Expression At baseline, there were no differences in 5-HTT protein expression (calculated as a ratio of 5-HTT to b-actin levels) between the hippocampal subregions (F 5 0.94, P > 0.05; n 5 6). There were also no baseline differences in 5-HTT protein expression in any of the subregions between the CORT treated mice and the vehicle treated controls (CA1: F 5 0.64, P > 0.05; CA3: F 5 0.59, P > 0.05; DG: F 5 0.81, P > 0.05, n 5 6/group). The vehicle treatment had no effect on 5-HTT protein expression in any hippocampal subregion compared with baseline (CA1: F 5 0.57, P > 0.05; CA3: F 5 0.74, P > 0.05; DG: F 5 0.68, P > 0.05, n 5 6/group). However, the CORT treatment decreased 5-HTT protein expression in the CA3 and the DG compared with the vehicle-treated controls (CA3: F 5 8.91, P < 0.05; DG: F 5 16.97, P < 0.05; n 5 6/group), but not in the CA1 (F 5 1.46, P > 0.05; n 5 6/group). The magnitude of the decrease in 5-HTT protein expression was significantly different between the CA3 and the DG (F 5 12.41, P < 0.05; n 5 6/group), with a 57% decrease in the DG compared with a 27% decrease in the CA3 (Fig. 4).

CORT Treatment Subregion-Specifically Decreased 5-HTT Function To determine whether 5-HTT function was also altered, we assayed [3H]5-HT uptake in synaptosomes, and then per-

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FIGURE 4. 5-HTT protein expression in the hippocampus following CORT treatment; 5-HTT protein expression was determined by Western Blot and presented as a ratio of 5-HTT to bactin levels. The vehicle treatment did not alter protein expression in any subregion of hippocampus (n 5 6/group; P > 0.05). However, the CORT treatment induced a significant decrease in protein expression in the CA3 and the DG (n 5 6/group). *P < 0.05 versus vehicle-treated controls; #P < 0.05 versus CORT-treated CA3 subregion by one-way ANOVA.

formed saturation kinetic analyses. At baseline, there were no differences in [3H]5-HT uptake as measured by Vmax or Km between the CA1, CA3, and DG subregions (Vmax: F 5 0.81, P > 0.05; Km: F 5 0.77, P > 0.05; n 5 6/group). ANOVA did not reveal any significant differences in Vmax (CA1: F 5 0.97, P > 0.05; CA3: F 5 0.99, P > 0.05; DG: F 5 0.87, P > 0.05; n 5 6/group) or in Km (CA1: F 5 1.01, P > 0.05; CA3: F 5 0.85, P > 0.05; DG: F 5 0.93, P > 0.05; n 5 6/ group) of [3H]5-HT uptake between the CORT treated mice and the vehicle-treated controls. The vehicle injection did not alter Vmax (CA1: F 5 0.76, P > 0.05; CA3: F 5 0.92, P > 0.05; DG: F 5 0.49, P > 0.05, n 5 6/group) and Km (CA1: F 5 0.96, P > 0.05; CA3: F 5 0.87, P > 0.05; DG: F 5 0.79, P > 0.05, n 5 6/group) of [3H]5-HT uptake in any sub-region. In contrast, the CORT treatment resulted in a decrease in the Vmax (F 5 6.97, P < 0.05; n 5 6/group) and an increase in the Km (F 5 7.01, P < 0.05; n 5 6/group) of [3H]5-HT uptake in the DG, but not in the CA1 (Vmax: F 5 0.96, P > 0.05; Km: F 5 1.07, P > 0.05; n 5 6/ group) or the CA3 (Vmax: F 5 1.02, P > 0.05; Km: F 5 0.76, P > 0.05; n 5 6/group), suggesting that depressive endophenotypes are associated with a lower total number of the membrane bound 5-HTT and a decreased 5-HT uptake efficiency in the DG (Fig. 5).

DISCUSSION Our study reports that hippocampal 5-HTT protein expression and function were decreased in CD-1 mice exhibiting endophenotypes of depression induced by chronic CORT Hippocampus

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FIGURE 5. Synaptosomal [3H]5-HT uptake in the hippocampus following CORT treatment. Saturation kinetic analyses of synaptosomal [3H]5-HT uptake were performed for each subregion of the hippocampus. Data were then fit to a Michaelis-Menten equation to derive Vmax and Km values. The vehicle treatment did not alter the saturation kinetics of [3H]5-HT uptake compared with the basal condition (n 5 6/group; P > 0.05). Following the CORT treatment, no changes in either measurement were found in the CA1 (A) or the CA3 (B), but a decrease in the Vmax and an increase in the Km were detected in the DG (C) compared with vehicle-treated controls (n 5 6/group). * P < 0.05 versus vehicletreated controls by one-way ANOVA.

treatment. Furthermore, the decrease in 5-HTT expression and function varied between subregions, with the DG showing the largest decrease, followed by the CA3, and no apparent change in the CA1. The depressive state was also associated with a decrease in tissue mass in the DG, but not in the CA1 or the CA3. The behavioral state of the CORT-treated mice was evaluated by sucrose preference test, physical state of the coat, FST and TST. Sucrose preference and coat state are commonly used to determine anhedonia in mice exposed to UCMS (Ducottet et al., 2003; Strekalova et al., 2006, 2011; Elizalde et al., 2008, 2010; Stremmelin et al., 2010; Tang et al, 2013). To our knowledge, the present study is the first to report a CORT-induced anhedonia in mice. FST and TST are often used to evaluate behavioral despair (David et al., 2009; Pollak et al., 2010; Yan et al., 2010; Naumenko et al., 2013). The decreases in immobility time following the CORT treatment Hippocampus

and the consistent lack of changes across weeks in control mice were in agreement with other studies using a similar CORT treatment regimen (Ago et al., 2008; Zhao et al., 2008). Thus, the three-week CORT treatment successfully induced anhedonia and behavioral despair, two core endophenotypes of clinical depression. In this study, 5-HTT protein expression, including both the membrane-bound and cytosolic 5-HTT levels, was decreased in the depressive state. Moreover, 5-HT saturation kinetics, which is a measure of the protein expression and functional activity of the membrane-bound 5-HTT, was altered in the depressive state as well (i.e., decreased Vmax and increased Km). Vmax is positively correlated with the amount of the membrane-bound 5-HTT, whereas Km is negatively correlated with the uptake efficiency of the membrane-bound 5-HTT. Hence, we concluded that both the total protein level and the membranebound level of 5-HTT were lower in the DG, and to a less extent in the CA3, compared with controls. Additionally, the functional activity of the membrane-bound 5-HTT, measured as the uptake efficiency, was reduced in a similar manner. Alterations of hippocampal 5-HTT associated with depression have been studied extensively in preclinical and clinical settings. Decreased hippocampal 5-HTT protein expression induced by the UCMS has been observed in the depressive state, which is then reversed by subsequent FLX treatment (Tang et al., 2013). Chronic social stress (CSS) models of depression also show decreased hippocampal 5-HTT binding (McKittrick et al., 2000). The current study showed consistent decreases in both protein expression and functional activity of hippocampal 5-HTT, which was induced by chronic CORT treatment. The UCMS, CSS, and the chronic CORT treatment are the three most commonly used rodent models of depression, all of which are based on the long-established connection between chronic stress and depression (Reus and Miner, 1985; Dinan, 1994; Heim and Binder, 2012). Taken together, these results suggest that decreases in hippocampal 5HTT protein expression and function are signature changes associated with stress-induced endophenotypes of depression. In contrast to our results, Zhu et al. (2010) reported that a single injection of lipopolysaccharide (LPS) enhanced hippocampal 5-HT uptake and increased immobility time in C57BL/6 mice. A single administration of LPS is not a typical method for inducing depression-related behavior, therefore, these results may be due to the acute effects of LPS on 5-HT uptake and behavior rather than to changes linked to depression. Discrepancies between studies may also be linked to mouse strain differences. Different mouse strains show differences in their responses to behavioral tests, in basal 5-HT levels and in responses to selective 5-HT reuptake inhibitors (SSRIs) (Lucki et al., 2001; David et al., 2003; Ripoll et al., 2003). Most recently, differences in 5-HTT expression were found to be associated with altered behavioral responses in the FST between mouse strains (Sugimoto et al., 2008). It is worth noting that chronic treatment with SSRIs has been shown to decrease 5-HTT expression and function in the hippocampus (Benmansour et al., 1999, 2002), which may

SUBREGION-SPECIFIC DECREASES IN HIPPOCAMPAL 5-HTT IN DEPRESSIVE STATE contribute to the therapeutic efficacy of SSRIs. This finding may seem at odds with the present results and others, as we found that depression-related endophenotypes correlated with decreased 5-HTT expression and activity. However, the rats used by Benmansour and colleagues were na€ıve to stress and showed no endophenotypes of depression. Therefore, these results could be due to the effects of SSRI’s on hippocampal 5-HTT under normal basal conditions rather than to changes related to the depressive state. A number of clinical studies have sought to measure 5-HTT changes both in the postmortem brain and recently in vivo using positron emission tomography (PET) in patients with depression. In agreement with our findings, several studies have reported decreases in 5HTT density (Leake et al., 1991; Arango et al., 1995), 5HTT binding (Joensuu et al., 2007), and 5-HTT immunoreactive axons (Austin et al., 2002) in various brain regions in depressed patients. PET studies also suggest that depression is linked to reduced 5-HTT binding in various brain regions, which was reversed as patients recovered (Ichimiya et al., 2002; Parsey et al., Mann JJ.; Bhagwagar et al, 2007; Selvaraj et al, 2011; Newberg et al., 2005, 2012). Although, the connection between lower 5-HTT function and depression is not universally observed (Staley et al., 2006; Savitz and Drevets, 2012), they raise the question of why patients with depression respond to SSRIs, which could lead to further decrease in 5HTT. Neuroadaptations may arise from chronic SSRI use to facilitate prior changes (Sharp and Cowen, 2011), but the more accepted neurobiological and physiological reasons why patients with depression respond to SSRIs remain a matter of debate. Direct interactions between CORT and 5-HTT have not been studied extensively, and the available evidence is mixed. Three-weeks of CORT treatment were shown to decrease hippocampal 5-HTT binding in Fisher rats (Maines et al., 1999), but there was no evaluation of depression-related endophenotypes. One-week of CORT ingestion did not alter hippocampal 5-HT uptake in both Spontaneously Hypertensive rats and Wistar-Kyoto rats (Fernandez et al., 2001). Moreover, in young Sprague-Dawley rats, there was no correlation between an acute CORT increase and hippocampal 5-HT uptake (Williams et al., 2005). A number of factors, including duration of exposure to CORT, dose and route of administration, could contribute to these different results. In addition, the rats used in these studies varied in their developmental stage, which could contribute to differences in sensitivity to the CORT treatment. Studies have shown that effects of CORT on hippocampal 5-HTT binding were age-dependent in rodents (Maines et al., 1999; Macri et al., 2009). Exposure to low levels of CORT in early developmental stages has been found to increase the production of auto-antibodies directed to 5-HTT in adulthood (Macri et al., 2009), which may subsequently decrease hippocampal 5-HT uptake. Furthermore, differences in basal hippocampal 5-HTT expression and function can differ across rat strains (Fernandez et al., 2003). Lastly, in addition to the 5HTT, hippocampal 5-HT uptake may occur through the organic cation transporter 3 (OCT3), which is also found to

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be regulated by CORT (Koepsell et al., 2007; Baganz et al, 2010). Although we can not rule out the possibility that the 5HT uptake mediated through OCT3 contributes to the overall 5-HT uptake measured in synaptosomes, future studies are needed to determine the 5-HT uptake mediated through 5HTT in the presence of a selective OCT3 antagonist in the depressive state. Furthermore, we found that the decreases in 5-HTT protein expression and function were subregion-specific. The DG showed the greatest decrease, which was also accompanied by a reduction in tissue mass. Recent studies have shown that chronic stresses, from psychosocial to physical, differentially influence neuronal morphology and physiology, and 5-HT transmission between hippocampal subregions (Joels, 2008). Chronic stress suppresses neurogenesis in the DG across different animal species and across different experimental paradigms (Mirescu and Gould, 2006; Joels et al., 2007; Joels, 2008). Although the underlying mechanisms are not known, it is recognized that stress increases CORT levels in the hippocampus (Keeney et al., 2006; Thoeringer et al., 2007; Droste et al., 2008), and that CORT in turn augments extracellular 5-HT in the hippocampus via glucocorticoid receptor (Summers et al., 2003; Barr and Forster, 2011). Hence, we speculate that the stress-induced rise in extracellular 5-HT may be mediated by negative feedback of CORT on 5-HTT in the DG. In summary, our results show that the chronic CORT treatment induced depressive endophenotypes that correlated with decreases in hippocampal 5-HTT protein expression and function. The DG is particularly sensitive to the effects of CORT, and most of the alterations in hippocampal 5-HTT, including the reduction in tissue mass, occurred in the DG. To help gain a more complete understanding of the pathophysiology of depression and its treatment, future studies should investigate whether similar functional changes in 5-HTT occur in vivo, and whether these changes occur in other critical brain regions, such as the amygdala, frontal cortex, and nucleus accumbens.

Acknowledgments The authors are thankful to Hongmei Li, Yang Li, Queliang Zhang, and Qing-hua Liu for their excellent technical assistance.

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