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Gene Dose-Dependent Alterations in Extraneuronal Serotonin but Not Dopamine in Mice with Reduced Serotonin Transporter Expression

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Tiffany A. Mathews, 1Denise E. Fedele, 1Francesca M. Coppelli, 3Amy M. Avila, 3Dennis L.

Murphy and 1,2Anne M. Andrews

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Department of Chemistry and 2Huck Institute for Life Sciences, The Pennsylvania State

University, University Park, PA 16802-6300.

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Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of

Health, 10 Center Drive MSC 1264, Bethesda, MD 20892-1264. Corresponding author: Anne Milasincic Andrews, 104 Chemistry Building, University Park, PA 16802-6300. E-mail: [email protected], TEL: 814-865-2970, FAX: 814-865-3314.

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Serotonin (5-HT) plays an integral regulatory role in mood, anxiety, cognition, appetite and aggressive behavior. Many therapeutic and illicit drugs that modulate these functions act at the serotonin transporter (SERT), thus a mouse model with reduced transporter expression was created to further investigate the effects of differential serotonin reuptake. In the present study, in vivo microdialysis was used to determine homeostatic alterations in extracellular 5-HT levels in unanesthetized SERT knockout mice. SERT-/- mice had significantly higher levels of basal dialysate 5-HT than SERT+/+ mice in striatum and frontal cortex. In addition, although genespecific increases in 5-HT were evident, neuroadaptive alterations in dialysate dopamine levels were not detected in striatum. Zero net flux microdialysis was utilized to further investigate alterations in extracellular 5-HT. Using this method, a gene dose-dependent increase in extraneuronal 5-HT was observed in striatum (2.8 ± 1, 9.4 ± 1 and 18 ± 3 nM) and frontal cortex (1.4 ± 0.4, 3.5 ± 0.9 and 14 ± 1 nM) in SERT+/+, SERT+/- and SERT-/- mice, respectively. Potassium stimulation revealed greater depolarization-induced increases in striatal 5-HT but not dopamine in SERT-/- mice. Furthermore, dialysate 5-HIAA levels were reduced in striatum in a gene dose-dependent manner, while DOPAC was unchanged in SERT knockout mice. Finally, determination of monoamine oxidase (MAO) activity revealed no significant differences in Km or Vmax of type-A or type-B isozymes indicating that alterations in SERT expression do not cause adaptive changes in the activities of these key catabolic enzymes. Overall, these results demonstrate that constitutive reductions in SERT are associated with increases in 5-HT in the extracellular signaling space in the absence of changes in dopamine neurochemistry. Furthermore, use of zero net flux microdialysis appears warranted in investigations of serotonergic synaptic function where modest changes in extracellular 5-HT are thought to occur in response to altered uptake.

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1. Introduction The serotonin (5-HT) neurotransmitter system is thought to play an important role in the regulation of mood, anxiety, cognition, appetite and aggressive behavior (Coccaro, 1990; Murphy, 1990; Murphy, 1999). Although there have been at least 14 different serotonin receptor subtypes identified so far, this system has a single high-affinity presynaptic plasma membrane transporter that actively clears serotonin from the extracellular space and is of central importance in the regulation of serotonergic signaling (Murphy, 1998; Pineyro, 1999; Gainetdinov, 2003). The significance of this transporter is further evident in the fact that many therapeutic agents (i.e., clomipramine, fluoxetine, citalopram, paroxetine) and illicit drugs (i.e., cocaine and 3,4methylenedioxymethamphetamine (MDMA)) act by inhibiting reuptake or causing reverse transport at the serotonin transporter (SERT). Many pre- and postsynaptic alterations have been shown to occur in response to chronic administration of serotonin reuptake inhibiting drugs (SRIs) (Kreiss, 1995; Nibuya, 1996; Hervas, 1998; Trillat, 1998; Gould, 1999; Pineyro, 1999; Zetterstrom, 1999; Duman, 2000; Malberg, 2000; Duman, 2001; Gardier, 2003; Santarelli, 2003). Together, these facets of neuroadaptation have led to the development of therapeutic models that relate changes in serotonergic neurotransmission to mood and anxiety disorders and their treatment. Furthermore, a functional polymorphism in the promoter region of the human SERT gene has been identified (Heils, 1996). This polymorphic element has "short" (s) and "long" (l) forms and individuals with the s/s or s/l genotypes exhibit reduced expression of SERT in brain, lymphoblasts and platelets (Lesch, 1996; Little, 1998; Greenberg, 1999). Moreover, decreased SERT expression in humans is associated with an increase in anxiety-related personality traits and susceptibility to stress-associated depression (Collier, 1996; Lesch, 1996; Greenberg, 1999; Caspi, 2003).

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To investigate directly the effects of constitutive reductions in SERT expression on neurochemistry, gene expression and behavior, mice genetically engineered to express reduced SERT have been generated using homologous recombination (Bengel, 1998). Since overt behavioral deficits, gross developmental abnormalities or increased mortality rates were not initially observed (Murphy, 2001), further studies have been conducted to identify more subtle aspects of an altered phenotype. Subsequently, behavior characterized by increased anxiety and fearfulness has been uncovered (Murphy, 2001; Holmes, 2003a; Holmes, 2003b; Holmes, 2003c). Biochemically, SERT knockout mice exhibit a gene dose-dependent decrease in SERT protein levels and 5-HT uptake, and a 60-80% reduction in total tissue 5-HT in mice lacking both copies of the SERT gene (Bengel, 1998; Montanez, 2003). Furthermore, constitutive reductions in SERT expression have been shown to have a significant impact on the expression and function of a number of serotonin receptor subtypes -- in particular 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C and 5-HT3 receptors (Li, 1999; Rioux, 1999; Fabre, 2000; Li, 2000; Liu, 2002; Li, 2003). Mice with decreased SERT expression also provide novel opportunities to further investigate the mechanisms of action of drugs that act at this molecular target. For example, MDMA-stimulated locomotor activity is attenuated in a gene dose-dependent manner in SERT knockout mice implicating SERT as a primary target for the stimulant effects of this recreationally abused substance (Bengel, 1998). Thus, the emerging picture of SERT knockout mice describes wide-ranging changes in the serotonin system and the behaviors it modulates; however, the extent to which extraneuronal 5-HT signaling is modified has not been fully ascertained. A previous study illustrated that SERT-/- mice show a 7-fold increase in basal dialysate 5-HT levels in substantia nigra compared to SERT+/+ mice (Fabre, 2000). Furthermore, a preliminary report suggested that 5-HT is

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increased in striatum in a gene dose-dependent manner (Fedele, 2001). In light of this, the foremost aim of the present study was to examine in greater depth changes in extracelluar 5-HT levels that accompany intermediate, as well as complete reductions in SERT expression. In vivo microdialysis was employed to measure extraneuronal 5-HT in striatum and frontal cortex. Since studies in mice (Bengel, 1998) and humans (Lesch, 1996; Greenberg, 2000; Caspi, 2003) suggest that intermediate reductions in SERT have a significant effect on behavior, we employed the method of zero net flux to determine specifically whether modest changes in extrasynaptic 5-HT levels occur in mice with a 50% reduction in SERT gene expression. Zero net flux is a quantitative microdialysis method that estimates extracellular neurotransmitter concentrations (Cext) by determining the point where there is no net diffusion of neurotransmitter between the extraneuronal space and the microdialysis membrane (Lonnroth, 1987; Bungay, 1990; Justice, 1993). This method also provides an approximation of "in vivo probe recovery". Strictly speaking, while there are no methods available to determine in vivo recovery experimentally, this parameter can be estimated by extraction fraction (Ed), which is the slope of the zero net flux regression line (Bungay, 1990; Bungay, 2003). Previous studies by Justice and co-workers have shown that pharmacologic inhibition of uptake alters extraction fraction (Justice, 1993; Olson, 1993; Cosford, 1994; Parsons, 1994; Olson Cosford, 1996). Thus, we hypothesized that alterations in transporter function driven by changes occurring at the level of gene expression would also influence extraction fraction. In addition to its effects on extracellular 5-HT, we assessed the consequences of constitutive decreases in SERT expression on striatal dialysate dopamine (DA) levels and K+stimulated 5-HT and DA release. We also examined dialysate levels of the 5-HT and DA metabolites, 5-hydroxyindoleacetic acid (5-HIAA) and 3,4-dihydroxytryptamine (DOPAC),

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respectively, as well as the possibility that alterations in 5-HIAA are associated with changes in monoamine oxidase (MAO) activity. The complete absence of SERT led to altered neurochemical homeostasis evidenced by increased 5-HT and decreased 5-HIAA levels in the extracellular signaling space. Intermediate reductions in SERT expression resulted in more subtle elevations in extraneuronal 5-HT that were detected only by zero net flux. On the other hand, the effects of reduced SERT expression appeared limited to the serotonergic system and did not affect dialysate DA and DOPAC levels in striatum or MAO-A and MAO-B activities.

2. Materials and Methods 2.1. Animals Male mice weighing at least 30 g were used for all experiments. Littermate mice expressing zero (SERT-/-), one (SERT+/-) or two (SERT+/+) intact copies of the SERT gene were generated as previously described on a mixed CD1 ¥ 129S6/SVev background (Bengel, 1998). A colony of SERT knockout mice was acquired via heterozygote matings and maintained at The Pennsylvania State University, where mice were housed in groups of 3-4 per cage with food and water ad libitum (12/12 hr light-dark cycle). Adolescent mice at the time of weaning (3-4 weeks old) were ear tagged and the terminal 2-3 mm of their tails were clipped for genotype identification by polymerase chain reaction (PCR) amplification of a region of exon 2 of the SERT gene.

2.2. Surgery Adult male mice were anesthetized with a combination of 48 mg/kg pentobarbital, 4.6 mg/kg xylazine and 0.04 mg/kg atropine, administered in a volume of 7-8 mL/kg, by the

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intraperitoneal (ip) route, to effect or in later experiments, with Avertin (Papaioannou, 1993) administered in a volume of 20 ml/kg, ip. The eyes were protected with sterile ophthalmic ointment during surgery (NLS Animal Health, Baltimore, MD). The skin over the skull was shaved, sterilized with Betadine and alcohol, incised and the exposed skull was cleaned and dehydrated with 10% H2O2. Mice were placed on a stereotaxic frame equipped with a mouse palate adapter and a burr hole was drilled (1 mm diameter). A guide cannula for a CMA/11 or, in later experiments, a CMA/7 microdialysis probe (CMA/Microdialysis, Chelmsford, MA) was implanted into the brain using coordinates determined from mouse brain atlases (Slotnick, 1975; Franklin, 1997) and refined by empirical determination (coordinates relative from Bregma for striatum: A+0.6, L-1.8, V-2.5 and frontal cortex: A+2.1, L-0.6, V-1.5). The guide cannula and surrounding exposed skull were sealed with a fast drying two-part epoxy (Locktite, Fastneal, State College, PA) that held the cannula in place. Mice were allowed to recover from surgery for 3-5 days prior to dialysis. After dialysis, mice were sacrificed by cervical dislocation and brains were removed for subsequent histological confirmation of probe placement. Experimental protocols strictly adhered to National Institutes of Health Animal Care guidelines and were approved by the Pennsylvania State University Institutional Animal Care and Use Committee.

2.3. Microdialysis The night before dialysis, mice were lightly anesthetized with 80 mg/kg ketamine and 10 mg/kg xylazine injected in a volume of 8 ml/kg, ip. The dialysis probe (CMA/11 or CMA/7, 2 mm length ¥ 240 mm diameter cuprophane, 6,000 MW cutoff) was inserted slowly and perfused overnight with artificial cerebrospinal fluid (aCSF) (concentrations in mM: 147 NaCl, 3.5 KCl, 1.0 CaCl2, 1.2 MgCl2, 1.0 NaH2PO4, 2.5 NaHCO3, pH 7.4, modified from (Trillat,

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1997)) at a rate of 1.1 mL/min. After a 10-hour equilibration period, six baseline samples were collected at 20-minute intervals and analyzed by online high performance liquid chromatography with electrochemical detection (HPLC-ED).

2.4. Chromatography Dialysate samples (20 ml) were injected immediately after collection onto an Advantage 100 ¥ 4.6 mm or in later experiments, a 100 ¥ 3.0 mm C18 ODS-II HPLC column (Thompson Instrument Co., Chantilly, VA) for separation followed by detection by an ESA 5014B microdialysis cell (E1 = -175 mV, E2 = +220 mV; ESA Coulochem II, ESA, Inc., Chelmsford, MA). A guard cell (ESA 5020) was placed in-line before the injection loop and set at a potential of +350 mV. The mobile phase consisted of 0.1 M monochloroacetic acid, 1.5-2.5 mM 1octanesulfonic acid, 5.5 ¥ 10-3 M ethylenediaminetetraacetic acid, 6-12% acetonitrile and 0.3% triethylamine in a volume of 1 L (pH 2.6) (Andrews, 1996). Characteristic retention times of DOPAC, DA, 5-HIAA and 5-HT were approximately 4, 5, 7 and 12 min, respectively. The neurotransmitter and metabolite peak areas were integrated using HP ChemStation software (Hewlett Packard, Waldbronn, Germany) and quantified against known standards. Concentrations are expressed in nM ± SEM. Limits of detection for 5-HT and DA were 0.1 nM and 0.6 nM, respectively (2 times signal to noise).

2.5. Zero net flux protocols At least six baseline samples were collected before the initiation of each experiment. SERT+/+, SERT+/- and SERT-/- mice were divided into three experimental groups: (1) striatal serotonin zero net flux with Cin concentrations bracketing Cext followed by stimulation with 120

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mM K+, (2) striatal serotonin zero net flux with high Cin concentrations to determine in vivo extraction fraction and (3) cortical serotonin zero net flux with Cin concentrations bracketing Cext. Experiments #1 and #3. After collection of baseline samples, four different concentrations of 5-HT ranging from 2-20 nM for SERT+/+ mice, 5-40 nM for SERT+/- mice and 10-50 nM for SERT-/- mice were perfused into the microdialysis probes for analyses conducted in striatum. Each concentration was perfused for 90 min using a programmable gradient infusion pump (CMA/Microdialysis, Chelmsford, MA). In frontal cortex, 5-HT zero net flux was performed essentially as described for striatum with the following modifications. Instead of having different infusion concentrations of 5-HT for each genotype, SERT+/+ and SERT+/- mice received the same concentrations (2-20 nM). In addition, the perfusion time for each concentration was increased from 90 to 100 minutes. In both striatum and frontal cortex prior to zero net flux, standards bracketing the expected dialysate 5-HT concentrations were analyzed. After assessing the resulting standard curves for linearity, aCSF containing the different Cin concentrations of 5-HT was pumped through the dialysis tubing. At this point in the experiment, the flow path bypassed the animal's head before going into the sample loop (for a more detailed discussion see Section 2.6). The resulting chromatograms were compared to standard curves to determine measured Cin concentrations, which were used for linear regression for zero net flux. Serotonin solutions for zero net flux were diluted immediately prior to use in aCSF containing 200 mM ascorbate as an antioxidant. Upon conclusion of zero net flux, at least 2 hours were allowed to pass for 5-HT levels to return to baseline before stimulated 5-HT release was investigated. For Experiment #1, mice were perfused with isoosmotic 120 mM K+ aCSF (in mM: 30.5 NaCl, 120 KCl, 1.0 CaCl2, 1.2 MgCl2, 1.0 NaH2PO4, 25 NaHCO3, pH 7.4, (Trillat, 1997)). K+-stimulation caused an

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increase in the 5-HT and DA peak areas differentiating them from non-biologically responsive substances in the chromatographic baseline. Experiment #2: A second zero net flux experiment was performed in striatum of SERT knockout mice to determine in vivo extraction fraction (slope of the zero net flux line) using high concentrations of Cin. Serotonin was perfused through the microdialysis probes at 100, 300, 500 and 700 nM in SERT+/+ mice and SERT-/- mice. Each concentration was randomly perfused for 100 min using a programmable gradient infusion pump (CMA/Microdialysis, Chelmsford, MA) and Cout was analyzed by HPLC-ED.

2.6. Measured in vivo recovery In the present experiments, we perfused Cin concentrations via a gradient perfusion pump through all of the dialysate tubing to a CMA/200 fraction collector/autosampler to determine the actual concentration of analyte entering the probe (Cin). When Cout was measured, perfusion syringes were filled with fresh solutions and a series of zero dead-volume liquid switches were moved to direct the flow path through the microdialysis probe and the animal's brain. The concentration of analyte exiting the probe (Cout) was subsequently determined. Since both Cin and Cout are measured, a more accurate linear regression plot can be constructed where Cin is plotted on the x-axis and the difference between Cin and Cout appears on the y-axis. The point where the line crosses the x-axis is referred to as Cext, which is the point where there is zero net diffusion across the microdialysis membrane. This point represents an estimate of the extracellular neurotransmitter concentration that corrects for submaximal recovery across the dialysis membrane.

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2.7. MAO-A enzyme assay Monoamine oxidase, present in the brain as well as other tissues, is a flavin-containing enzyme that catalyzes the oxidation of primary amines to produce the analogous aldehydes, H2O2 and ammonia according to the following general reaction (Youdim, 1987): RCH2NH2 + O2 + H2O Æ RCHO + NH3 +H2O2 MAO is found in two isoforms, MAO type-A and type-B, which can be differentiated based on specificities for different substrates and inhibitors. Serotonin, dopamine and norepinephrine are substrates for MAO-A (Shih, 1999). DA also is oxidized by MAO-B. MAO-A activity was determined by measuring the rate of conversion of tryptamine (TRP) to indole-3-acetic acid (I-3AA) using HPLC-ED at +0.8 V (BAS LC-4C, West Lafayette, IN) (Andrews, 1996). Crude mitochondrial fractions were prepared from the brains of SERT+/+, SERT+/- and SERT-/- mice by homogenization of the tissue in cold 0.32 M sucrose (10 mL/g original wet weight) using a motor-driven Teflon pestle followed by centrifugation at 12,000 g for 10 minutes at 4 °C. The resulting pellets were resuspended in the same volume of cold 0.32 M sucrose and centrifuged at 21,000 g for 20 minutes at 4 °C. Tissue then was suspended in 0.1 M phosphate buffer (20 mL/g original wet weight), pH 7.4, centrifuged at 12,000 g for 10 minutes at 4 °C, decanted and stored at ­70 °C. On the day of analysis, brain tissue was homogenized briefly in assay buffer containing 0.1 M phosphate, pH 7.4, and 3 mM nicotinamide adenine dinucleotide (NAD) (30 ml/g original wet weight). Six hundred ml aliquots of tissue were preincubated with 0.25 mM selegiline for 15 minutes at 37 °C to irreversibly inhibit MAO-B (Sonsalla, 1987). Following preincubation, varying concentrations of tryptamine were added to samples in duplicate (4-50 mM). In addition,

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alcohol dehydrogenase (0.13 U/mL) was added to convert the intermediate product, indole-3acetaldehyde, to an electrochemically active final product, I-3AA, in the presence of NAD. Samples were incubated at 37 °C for 30 minutes in a final volume of 1 mL after which they were extracted with 250 mL of 0.5 M HClO4 and centrifuged at 7200 g for 10 minutes. Fifty mL of each supernatant was injected onto a 10 cm ¥ 4.6 mm Advantage 3 mm ODS reversed-phase chromatography column (Thomson Instruments, Springfield, VA) in a mobile phase containing 0.1 M monochloroacetic acid, 0.55 g/L octanesulfonic acid, 0.3% triethylamine, 25% acetonitrile and 10 mM EDTA, pH 2.6 at a flow rate of 0.5 mL/min. Protein was determined by the method of Lowry et al. (Lowry, 1951).

2.8. MAO-B enzyme assay MAO-B activity was measured using the Amplex Red Monoamine Oxidase Assay Kit (Zhou, 1997). As shown above, one of the byproducts of the oxidation of primary amines by MAO is H2O2, which can be quantified as a measure of MAO activity. We analyzed MAO-B activity using a horseradish peroxidase (HRP) reaction with the nonfluorescent reagent N-acetyl3,7-dihydroxyphenoxazine, also known as Amplex Red. Reduction of H2O2 by HRP is coupled to the oxidation of Amplex Red to yield the fluorescent product resorufin. Amplex Red reacts with H2O2 in a 1:1 ratio and the resulting fluorescence can be used to determine the concentration of H2O2 produced by MAO-B catalyzed oxidation of the substrate benzylamine (BAM). Crude mitochondrial fractions were prepared from the cortex of SERT+/+, SERT+/- and SERT-/- mice as described in Section 2.7. MAO-B analysis was carried out as per the manufacturer's instructions. Tissue samples were thawed and diluted to a final protein

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concentration of 8 mg/mL and MAO-A was inhibited with 1 mM clorgyline. Following preincubation with clorgyline at room temperature for 30 min, varying concentrations of benzylamine were added to samples in triplicate (10-550 mM). Fluorescence was determined at 490/590 nm. Product amounts were calculated using linear regression equations obtained from resorufin standard curves (0-20 mM) to determine the concentration of H2O2 evolved. Protein was determined by the method of Lowry et al. (Lowry, 1951).

2.9. Chemicals and drugs Components of HPLC mobile phases, artificial cerebrospinal fluid and neurotransmitter standards were of HPLC grade or the highest quality obtainable from Sigma-Aldrich (St. Louis, MO). Selegiline was from Chinoin Pharmaceutical Works (Budapest, Hungary). The Amplex Red Monoamine Oxidase Assay Kit containing all chemicals necessary for the MAO-B assay was purchased from Molecular Probes, Inc. (Eugene, OR; #A-12214). All other components for the MAO-A and MAO-B assays were prepared with materials from Sigma-Aldrich or a comparable source.

2.10. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) with genotype as the independent variable or two-way ANOVA with time as the repeated measure using the Statistical Analysis System (SAS Institute, Carey, NC). Zero net flux data were analyzed by linear regression to generate x-intercepts (Cext) and slopes (Ed) for individual animals. MAO data were analyzed by linear regression of double reciprocal Lineweaver-Burke plots to generate y-intercepts (1/Vmax) and slopes (KM/Vmax) for trials on individual days with respect to genotype. A priori

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significant differences between genotypes are indicated by t-test probabilities. All values are expressed as means ± SEM, with differences of p<0.05 considered statistically significant. Significance is denoted in the figures as: ***p<0.001, **p<0.01, *p<0.05 versus appropriate control groups. Duncan's post hoc comparisons between SERT+/- mice and SERT-/- mice are indicated by ap <0.05.

3. Results 3.1. Basal dialysate 5-HT and DA levels in striatum in SERT knockout mice To determine whether life-long reductions in SERT expression influence the concentration of serotonin in the extracellular milieu, dialysate 5-HT levels were determined in striatum and averaged over six baseline samples per mouse from a total of 29 mice. Analysis of variance revealed a significant difference between mean dialysate 5-HT concentrations with respect to genotype [F(2,26)=4.72; p<0.05] (Fig. 1A). Dialysate 5-HT in SERT+/+ mice was 0.32 ± 0.06 nM, which did not differ significantly from mean 5-HT levels in SERT+/- mice (0.32 ± 0.05 nM). On the other hand, basal dialysate 5-HT was significantly increased to 2.6 ± 0.9 nM in striatum in SERT-/- mice. Since the serotonin and dopamine neurotransmitter systems are thought to act together to modulate locomotor activity and reward-related behavior (Gudelsky, 1996; Sora, 2001), we determined whether reductions in SERT expression exert a neuroadaptive effect on extracellular DA. In striatum, dialysate DA concentrations averaged over six baseline samples collected from 17 mice showed no significant differences with respect to genotype [F(2,14)=0.78; p=0.5] (Fig. 1B). Mean dialysate DA levels were 2.4 ± 0.4 nM in SERT+/+ mice, 3.1 ± 1 nM in SERT+/- mice and 4.2 ± 1 nM in SERT-/- mice. Together, these data suggest that decreased SERT expression

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has a selective effect to increase dialysate 5-HT versus DA in mice with a complete loss of SERT.

3.2. K+-stimulated 5-HT and DA efflux The effect of constitutive reductions in SERT expression on serotonin and dopamine release also were investigated using microdialysis. Administration of 120 mM K+ for 20 minutes via the microdialysis probe elevated dialysate 5-HT during a single 20-minute sample (Fig. 1A). The univariate genotype ¥ time interaction was not significant [F(12,156)=3.05; p=0.064] indicating that high K+ had a similar effect on 5-HT with respect to genotype. The main effect of time was significant [F(6,156)=11.96; p<0.01] demonstrating that high K+ significantly stimulated 5-HT efflux in all genotypes. K+-stimulated 5-HT levels were significantly different with respect to genotype [F(2,29)=4.09; p<0.05]. Striatal 5-HT levels were increased to 2.1 ± 0.3 nM in SERT+/+ mice and this was not different from the level observed in SERT+/- mice (3.2 ± 0.6 nM). By contrast, SERT-/- mice showed a significantly greater increase in K+-stimulated extracellular 5-HT in striatum to 9.7 ± 3 nM with respect to SERT+/+ mice (p<0.01). Perfusion of high K+ also increased extracellular DA during the subsequent 20-minute dialysate sample (Fig. 1B). In this case, the genotype ¥ time interaction also was not significant [F(12,60)=0.47; p=0.6] showing that DA levels were similar across time in the three genotypes of mice. The main effect of time was significant [F(6,60)=16.8; p<0.01] demonstrating that high K+ significantly increased dialysate DA. However similar to basal DA levels, no significant differences in dialysate DA in response to 120 mM K+ were observed with respect to genotype [F(2,10)=0.22; p=0.8]. Striatal DA levels were increased to 29 ± 7 nM in SERT+/+ mice, 39 ± 20 nM in SERT+/- mice and 25 ± 9 nM in SERT-/- mice following high K+. These data indicate that

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complete loss of SERT potentiates the stimulated concentration of 5-HT but not DA in the extraneuronal signaling space.

3.3. Zero net flux determination of extracellular 5-HT in SERT knockout mice - striatum Previous studies in SERT deficient mice have revealed a gene dose-dependent deficit in SERT expression, 5-HT uptake and MDMA-stimulated locomotor activity (Bengel, 1998; Montanez, 2003). Determination of dialysate 5-HT levels without correction for extraction fraction, however, did not indicate an increase in extracellular 5-HT levels in SERT+/- mice (Figs. 1A and 2B). If serotonin uptake is decreased in mice expressing one functional copy of the SERT gene as evidenced by the intermediate phenotype observed after MDMA, then we hypothesized that basal 5-HT levels would be modestly elevated in SERT+/- mice. To test this hypothesis, we employed the method of zero net flux. Concentrations of 5-HT flowing into and out of the microdialysis probes were determined and plots of Cin versus Cin­Cout were constructed to estimate extracellular 5-HT levels (Cext, x-intercept) and extraction fractions (Ed, slope) (Lonnroth, 1987; Justice, 1993; Olson, 1993). Using this method, significant differences in the extracellular concentration of 5-HT with respect to genotype in striatum were evident [F(2,23)=12.9; p<0.001] (Figs. 2A and 2B). Extracellular serotonin levels estimated by zero net flux were 2.8 ± 1 nM in SERT+/+ mice. In SERT-/- mice, striatal 5-HT was 18 ± 3 nM, which was significantly higher than the level in SERT+/+ mice (p<0.01). Furthermore, extracellular 5-HT in SERT+/- mice was 9.4 ± 1 nM, and this level was significantly greater than that in SERT+/+ mice (p<0.05). Post hoc analysis revealed that extracellular 5-HT also was significantly different between SERT+/- and SERT-/mice (p<0.05).

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By contrast, extraction fraction was not significantly different across genotypes [F(2,23)=0.56; p=0.6] (Figs. 2A and 2B). SERT+/+ mice showed an extraction fraction of 0.37 ± 0.08, while SERT+/- mice had an Ed of 0.32 ± 0.04 and in SERT-/- mice, Ed was 0.30 ± 0.01. Overall, the method of zero net flux yields data in support of the hypothesis that intermediate reductions in SERT expression lead to measurable increases in basal 5-HT in striatum, however, they do not provide evidence for altered in vivo extraction fraction.

3.4. Estimation of in vivo extraction fraction using high concentrations of Cin in striatum In a previous study, Olson Cosford et al. used zero net flux to determine extracellular 5HT in the nucleus accumbens in rats and showed that changes in in vivo extraction fraction are better determined using high concentrations of Cin where changes in slope are more apparent (Olson Cosford, 1996). Thus, 100, 300, 500 and 700 nM 5-HT was perfused into striatum in SERT+/+ and SERT-/- mice to investigate further whether reduced transporter function could be detected as a decrease in the slope of the zero net flux line. Even using these concentrations of Cin, however, zero net flux analysis revealed no significant differences in in vivo extraction fraction [F(1,13)=0.13; p=0.7] (Fig. 3). Under these conditions, SERT+/+ mice showed an average extraction fraction of 0.24 ± 0.05, while SERT-/- mice had an average Ed of 0.27 ± 0.08.

3.5. Extracellular 5-HT without correction for in vivo extraction fraction in frontal cortex In addition to striatum, the serotonin system projects to other brain regions including most areas of the cortex. Serotonergic innervation to frontal cortex has been implicated in the modulation of mood and anxiety-related behavior, and in the mechanism of action of antidepressants (Gobert, 1997; Invernizzi, 1997; Sharp, 1997; Gardier, 2003); therefore, we

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investigated the effects of constitutive decreases in SERT expression on basal serotonin levels in this brain region. Dialysate 5-HT levels were determined in frontal cortex and averaged over six baseline samples per mouse from a total of 15 animals (Fig. 4B). In SERT+/+ mice, dialysate 5HT in frontal cortex was 1.0 ± 0.07 nM and this did not differ significantly from the level measured in SERT+/- mice (1.2 ± 0.2 nM). However, analysis of variance indicated that a significant difference was present in cortical dialysate 5-HT with respect to genotype [F(2,16)=7.96; p<0.01] and that this was due to increased dialysate levels of 5-HT in SERT-/mice (3.6 ± 0.5 nM; p<0.01 vs. SERT+/+ and p<0.05 SERT+/- mice). These results further demonstrate that SERT is a primary regulator of 5-HT in the extraneuronal signaling space in frontal cortex.

3.6. Zero net flux determination of 5-HT in SERT knockout mice ­ frontal cortex Zero net flux also was employed to assess extraneuronal serotonin concentrations in frontal cortex because in striatum, increases in 5-HT levels in SERT+/- mice were only detectable using this method of analysis. One-way ANOVA indicated a significant difference in extracellular concentrations of 5-HT with respect to genotype [F(2,12)=56.6; p<0.001] (Figs. 4A and 4B). Cortical 5-HT determined by zero net flux in SERT+/+ mice was 1.4 ± 0.4 nM. In SERT-/- mice, cortical 5-HT was 14 ± 1 nM, which was significantly higher than the level in SERT+/+ mice (p <0.001). Extracellular 5-HT in SERT+/- mice showed a trend towards an increase, however this did not reach statistical significance (3.5 ± 0.9 nM; p=0.1). Unlike in striatum, significant differences in extraction fraction were observed in frontal cortex [F(2,12)=6.41; p<0.01] (Fig. 4B). SERT+/+ mice had an average extraction fraction of 0.51 ± 0.05, while SERT+/- mice showed a trend towards decreased Ed (0.39 ± 0.04; p=0.1) and in

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SERT-/- mice, Ed was 0.27 ± 0.05 (p<0.01 vs. SERT+/+ mice). These data demonstrate that loss of SERT expression leads to a significant increase in extracellular 5-HT levels in frontal cortex, as well as striatum. In addition, while not statistically significant, they point to a trend towards modestly increased levels of extracellular 5-HT in frontal cortex in mice lacking one copy of the SERT gene.

3.7. Basal dialysate concentrations of 5-HIAA and DOPAC in striatum We determined whether decreases in SERT expression result in alterations in extraneuronal serotonin and dopamine metabolite concentrations. One-way ANOVA demonstrated that 5-HIAA levels analyzed from a total of 42 mice were significantly different with respect to genotype [F(2,39)=7.74; p<0.001] (Fig. 5). Extracellular 5-HIAA in SERT+/+ mice was 200 ± 20 nM. In SERT+/- mice, mean 5-HIAA levels were decreased to 150 ± 10 nM (p<0.01 vs. SERT+/+ mice). The average concentration of 5-HIAA in SERT-/- mice was determined to be 120 ± 10 nM (p<0.001 vs. SERT+/+ mice). On the other hand, striatal DOPAC levels analyzed in 29 mice showed no significant differences with respect to genotype [F(2,26)=0.12; p=0.9] (Fig. 4). Dialysate DOPAC analyzed from SERT+/+, SERT+/- and SERT-/- mice was 910 ± 200 nM, 940 ± 200 nM and 1000 ± 200 nM, respectively. Thus far, the results indicate that reduced SERT expression is associated with gene dose-dependent increases in extracellular 5-HT levels and decreases in 5-HIAA levels, though no changes in dialysate levels of DA or its metabolite DOPAC are evident in SERT knockout mice.

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3.8. MAO activity in SERT knockout mice Serotonergic neurons primarily contain MAO-B (Levitt, 1982). Since reduced amounts of 5-HT are taken up into presynaptic serotonin neurons in SERT knockout mice, particularly in SERT-/- mice, we assessed whether decreased SERT expression exerts a neuroadaptive effect on MAO-B activity. Using benzylamine as a substrate in brain mitochondrial preparations, no statistically significant differences in the KM [F(2,6)=0.16; p=0.9] or Vmax [F(2,6)=1.5; p=0.3] of MAO-B were detected with respect to genotype (Fig. 6). KM was 55 ± 10, 62 ± 3 and 54 ± 10 mM and Vmax was 9.0 ± 1.6, 12 ± 0.8 and 11 ± 1 nmol/mg protein/hr in SERT+/+, SERT+/- and SERT-/- mice, respectively. Although serotonin neurons express MAO-B, 5-HT is a preferred substrate for MAO-A. Others have shown that 5-HT can be taken up by dopaminergic and noradrenergic neurons under conditions of decreased 5-HT reuptake in vivo (Daws, 1998; Pan, 2001; Zhou, 2002) and catecholaminergic neurons contain MAO-A (Westlund, 1985; Kitahama, 1986). Thus, we analyzed the activity of this MAO isozyme to determine whether decreased MAO-A activity might account for diminished 5-HIAA levels in SERT knockout mice. As was the case for MAO-B, no statistically significant differences were seen in either the KM [F(2,6)=0.31; p=0.7] or Vmax [F(2,6)=0.04; p=1] of MAO-A using tryptamine as a substrate. For MAO-A, KM was 2.2 ± 0.4, 2.5 ± 0.2 and 2.5 ± 0.3 mM and Vmax was 3.2 ± 0.6, 2.9 ± 0.5 and 3.0 ± 0.9 nmol/mg protein/hr in SERT+/+, SERT+/- and SERT-/- mice, respectively. Together, these data show that neither MAO-A nor MAO-B activity is altered in mice with reduced SERT expression.

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4. Discussion Mice with constitutive reductions in serotonin transporter expression have been generated to elucidate further the role of SERT in normal behavior, disease processes and the mechanisms of action of numerous drugs that interact with this transporter (Bengel, 1998; Murphy, 2003). A number of phenotypic alterations have been discovered to occur in response to the complete absence of SERT including increased anxiety-like behavior, decreased aggressive behavior, regional alterations in 5-HT receptor subtype expression and function, and differential responses to cocaine and the substituted amphetamine MDMA (Bengel, 1998; Sora, 1998; Li, 1999; Rioux, 1999; Fabre, 2000; Li, 2000; Holmes, 2002; Holmes, 2003c; Li, 2003). Serotonergic neurons show decreased firing rates most likely mediated by desensitization of presynaptic 5-HT1A receptors in SERT+/- and SERT-/- mice (Gobbi, 2001). In addition, prolonged recovery of firing rates in postsynaptic hippocampal neurons after stimulation with 5-HT is associated with decreased sensitivity of postsynaptic 5-HT1A receptors in SERT-/- mice. Downregulation of enteric 5-HT3B receptor subunits has been reported to affect gut motility in SERT-/- mice (Liu, 2002). Additionally, mice lacking SERT display increased rapid eye movement sleep and exaggerated adrenomedullary responses to stress (Tjurmina, 2002; Armando, 2003; Wisor, 2003). Most importantly, all of these biochemical and behavioral alterations in mice with reduced SERT expression are hypothesized to result directly or indirectly from elevated extraneuronal 5-HT levels, since the primary function of SERT is to clear 5-HT from the extracellular space. Thus, the major goal of this study was to determine whether life-long reductions in SERT expression cause elevations in extracellular 5-HT levels that can be detected using the technique of in vivo microdialysis.

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A previous microdialysis study without correction for in vivo extraction fraction showed that SERT-/- mice have elevated dialysate 5-HT levels in substantia nigra compared to SERT+/+ mice (Fabre, 2000). In the present study, we demonstrate that SERT-/- mice have increased dialysate 5-HT levels in striatum and frontal cortex as well. Our data also indicate that basal dialysate 5-HT levels determined without correction for extraction fraction are not significantly different between SERT+/+ and SERT+/- mice in both brain regions studied. However, other evidence suggests that intermediate reductions in SERT expression are not without effect. Determination of uptake by high-speed chronoamperometry in the CA3 region of the hippocampus indicates that the rate of 5-HT clearance from the extracellular space is decreased in SERT+/- mice (Montanez, 2003). SERT+/- mice also are more sensitive to 5-HT uptake inhibition resulting in longer clearance times for serotonin in response to fluvoxamine. SERT+/mice exhibit downregulation and desensitization of 5-HT1A receptors in the dorsal raphe, while 5-HT1B receptors are downregulated in the nucleus accumbens and substantia nigra (Fabre, 2000; Li, 2000; Gobbi, 2001). In addition, SERT+/- mice exhibit an intermediate response to the locomotor-stimulating effects of MDMA compared to SERT+/+ and SERT-/- mice (Bengel, 1998). Moreover, humans with a similar level of reduced SERT expression have been characterized by increased negative emotionality and anxiety-related personality traits (Lesch, 1996; Greenberg, 2000), and they appear to be more susceptible to depression in response to stressful life events (Caspi, 2003). Thus, the combined data from mice and humans support the hypothesis that intermediate reductions in SERT expression result in modest changes in 5-HT neurotransmission. In order to determine whether subtle but biologically significant alterations in extracellular 5-HT occur in SERT+/- mice, the method of zero net flux was employed. This

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method of analysis takes into account differences in the exchange of 5-HT between the dialysis membrane and the surrounding tissue in the form of in vivo extraction fraction. The latter is thought to be sensitive to changes in active processes, particularly uptake by plasma membrane transporters (Justice, 1993). In the present case, data obtained by zero net flux in striatum and frontal cortex agree with those without correction for extraction fraction showing that SERT-/mice have elevated extracellular 5-HT levels. However, zero net flux analysis also illustrates that extracellular 5-HT concentrations are underestimated by basal dialysate measurements (2.8 versus 0.3 nM and 18 versus 2.6 nM in striatum in SERT+/+ and SERT-/- mice, respectively). Notably, zero net flux also revealed an increase in striatal and cortical concentrations of extracellular 5-HT in SERT+/- mice, even though 5-HT levels were elevated only modestly compared to SERT+/+ mice, especially in the case of frontal cortex (3-fold increase in striatum and 2-fold in frontal cortex). Therefore, under conditions of altered transport, in vivo microdialysis in combination with zero net flux can expose small but important differences in extracellular neurotransmitter concentrations that cannot be resolved using standard microdialysis methods alone. Similar findings have been reported in mice with partial reductions in dopamine transporter expression (Jones, 1998). Mice with intermediate reductions in norepinephrine transporter expression have not been studied in this regard. Justice and coworkers have shown that in vivo extraction fraction determined by the slope of the zero net flux regression line is correlated with the function of plasma monoamine transporters and that administration of uptake inhibitors decreases extraction fraction (Parsons, 1991; Parsons, 1992; Justice, 1993; Parsons, 1993; Parsons, 1994; Olson Cosford, 1996). Thus, we hypothesized that SERT knockout mice would exhibit a gene dose-dependent reduction in in vivo extraction fraction. Using low concentrations of Cin, a weak trend toward decreased Ed with

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respect to genotype was observed in striatum but the differences were not statistically significant. However, Justice and colleagues determined extraction fraction using high 5-HT concentrations (100-700 nM) perfused into the nucleus accumbens (Olson Cosford, 1996). They reasoned that differences in slope were more apparent at high Cin concentrations, while low Cin concentrations were better for determining Cext (Olson Cosford, 1996). Thus, we further hypothesized that extraction fraction in SERT knockout mice using low Cin concentrations (2.5­50 nM) was not optimally determined. Even when high concentrations of 5-HT were infused into striatum in SERT knockout mice, no statistically significant differences continued to be observed for striatal in vivo extraction fraction. By contrast, low concentrations of Cin perfused into frontal cortex elucidated a significant decrease in Ed with respect to genotype. Since in vivo extraction fraction in frontal cortex was sensitive to reductions in 5-HT reuptake, tissue factors appear to dominate resistance to diffusion in this brain region (Tang, 2003). However, Ed is relatively insensitive to changes in reuptake when its magnitude is largely determined by diffusional resistance through the dialysis membrane, which may be the case in striatum. Data from striatum suggest that SERT may play a secondary or indirect role relative to other mechanisms of 5-HT clearance in this brain region. Future studies aimed at comparing in vitro extraction fraction under well-stirred conditions to in vivo extraction fraction in striatum versus frontal cortex in mice will be important for determining the relative contributions of diffusional resistance due to the tissue versus the dialysis probe in these two brain regions. As the case may be, the responsiveness of in vivo extraction fraction in different brain regions to constitutive reductions in 5-HT reuptake suggests that local environments with differing axon and transporter densities may contribute to the regional variability in the behavior of in vivo extraction fraction in SERT knockout mice.

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Regional variations in innervation and uptake site density in prefrontal cortex versus striatum in the dopamine neurotransmitter system have been shown to lead to differences in the diffusion characteristics of dopamine (Garris, 1993; Garris, 1994; Cass, 1995; Sesack, 1998). While quantitative microdialysis is the only method available at present to assess basal extracellular monoamine neurotransmitter concentrations, a number of disadvantages have been attributed to this technique including damage to brain tissue associated with the relatively large diameter of the microdialysis probe and low temporal resolution. Sampling times on the order of minutes have most often been reported for microdialysis studies, however, Kennedy and coworkers recently described sampling intervals as small as 10 s for the determination of amino acid neurotransmitters (Boyd, 2000). Therefore, reductions in sampling times are possible using small diameter capillaries and electrophoresis or narrow bore liquid chromatography column separations (McKenzie, 2002). With regard to tissue damage, neuronal density is reportedly decreased at least 400 mm away from the microdialysis probe tract and intracellular disruption was observed as far as 1.4 mm from the probe (Clapp-Lilly, 1999). Recently, tissue damage caused by the dialysis probe has spurred debate over the effectiveness of microdialysis to accurately estimate concentrations of biological molecules sampled from the brain extracellular space (Peters and Michael, 1998; Bungay et al., 2003). Most investigators agree however, that neurotransmitter levels determined without some type of estimation of probe recovery underestimate actual extracellular neurotransmitter concentrations in the brain. Furthermore, the results of this study, as well as those utilizing dopamine transporter knockout mice demonstrate that differential extracellular neurotransmitter levels are detected even under conditions of intermediate reductions in transporter expression using zero net flux microdialysis methods (Jones, 1998).

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5-Hydroxyindoleacetic acid is the primary metabolite of serotonin resulting from enzymatic breakdown of 5-HT by monoamine oxidase. In this study, a gene dose-dependent decrease in extracellular 5-HIAA was observed in SERT knockout mice. This may reflect reduced uptake and subsequent intraneuronal degradation of 5-HT in serotonergic neurons. However, substantial 5-HIAA levels were still present in SERT-/- mice indicating that conversion of 5-HT to 5-HIAA by monoamine oxidase occurs even in the absence of SERT. Possible sources for 5-HIAA in SERT-/- mice include newly synthesized 5-HT, which is subject to catabolism by intracellular MAO prior to sequestration by the vesicular monoamine transporter (Jones, 1998). In addition, 5-HT may be taken up from the extracellular space by other plasma membrane transporters. For instance, dopamine and norepinephrine transporters have been shown to transport 5-HT, particularly under conditions of reduced 5-HT reuptake (Cases, 1998; Daws, 1998; Pan, 2001; Zhou, 2002) and once inside dopaminergic or noradrenergic neurons, 5HT can be degraded by MAO. Mice lacking MAO-B, the predominant form of MAO expressed in serotonergic neurons show normal levels of 5-HIAA providing further evidence for production of 5-HIAA in extra-serotonergic locations (Levitt, 1982; Kitahama, 1986; Kitahama, 1994; Grimsby, 1997). In fact, only mice lacking the MAO-A isoform not expressed in serotonergic neurons exhibit reductions in 5-HIAA levels (Cases, 1995). With regard to SERT knockout mice, decreases in 5-HIAA do not appear to be attributable to alterations in the activity of MAO-A or MAO-B. Furthermore, gene dose-dependent decreases in extracellular 5-HIAA were detected without correction for in vivo extraction fraction. This is in keeping with the fact that 5-HIAA is not a substrate for active transport (Parsons, 1992). To induce nontransporter-mediated release of 5-HT, 120 mM K+ was perfused directly into striatum causing neuronal depolarization and exocytotic release of neurotransmitters.

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Although increases in striatal 5-HT levels were observed in all genotypes of SERT knockout mice, K+-stimulated 5-HT levels in SERT-/- mice were significantly higher than those in SERT+/+ mice. Serotonin levels following infusion of high concentration K+ most likely reflect the sum of the release and uptake processes under the current conditions; therefore, while increases in K+stimulated 5-HT levels in SERT-/- mice are thought to result from the absence of 5-HT reuptake, increased release cannot be ruled out as contributing to this observation. However, Blier and colleagues have demonstrated that serotonergic firing rates show compensatory reductions in response to decreased SERT expression leading to decreased stimulation-induced 5-HT release (Gobbi, 2001). Prolonged neurotransmitter clearance, changes in pre- and postsynaptic homeostasis and a range of phenotypic alterations in behavior, some of which were unanticipated, are hallmarks of mice genetically engineered to lack the monoamine transporters (Gainetdinov, 1999; Murphy, 2001; Gainetdinov, 2003; Torres, 2003). In all cases, elevations in extracellular monoamine levels are of central importance to these neuroadaptive effects. In mice lacking the dopamine transporter, a 5-fold increase in extracellular DA levels in striatum has been demonstrated (Giros, 1996; Jones, 1998). By contrast, mice with constitutive loss of NET show a more modest 2-fold increase in extracellular norepinephrine in cerebellum (Xu, 2000). The present results, demonstrate that SERT-/- mice exhibit significant 6- and 10-fold increases in extracellular 5-HT levels in striatum and frontal cortex, respectively. Notably, these data also reveal that SERT+/mice show smaller magnitude increases in extraneuronal 5-HT; however, these increases in extracellular 5-HT levels were detected only by the method of zero net flux. Thus, elevated extracellular serotonin levels even under conditions of moderate reductions in SERT expression appear to modulate 5-HT receptor expression and/or function in SERT knockout mice (Li, 1999;

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Rioux, 1999; Fabre, 2000; Li, 2000; Liu, 2002; Li, 2003) and presumably, in humans so as to underlie key aspects of altered behavior including increased anxiety-related behavior (Lesch, 1996; Greenberg, 2000; Holmes, 2003a; Holmes, 2003b) and in humans, increased susceptibility to major depressive episode in relation to stressful life events (Caspi, 2003).

Acknowledgements: The authors wish to express sincere gratitude to Angela C. Wolford and Dawn E. Rupp for their technical expertise performing the microdialysis experiments and to Erin J. Steck for her assistance with the MAO-B assay development. They are also grateful to Erica L. Unger for managing the SERT knockout mouse breeding colony and to Laura E. Killingbeck for assistance with the manuscript. Funding was providing by the Pennsylvania State University Department of Chemistry, the National Institute of Mental Health (5R03MH0677) and Eli Lilly and Company.

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34

A.

14 12 10 8 6 4 2 0 -100

SERT+/+ SERT+/SERT-/-

**

120mM K+

-80

-60

-40

-20

0

20

Time (mins)

B.

60 50 40 30 20 10 0 -100

SERT+/+ SERT+/SERT-/-

120mM K+

-80

-60

-40

-20

0

20

Time (mins)

Figure 1

35

Figure 1: (A) Basal dialysate 5-HT levels (n = 10, 7 and 12, respectively) and (B) DA levels (n = 6, 6 and 5, respectively) in striatum in SERT+/+, SERT+/- and SERT-/- mice. Between times T-20 and T0 120 mM K+ was perfused through the microdialysis probes to stimulate neurotransmitter release.

36

A.

15 10 5 0 10 -5 20 30 [5-HT] in 40 50 SERT+/+ SERT+/SERT-/-

B.

50 40 30 20 10 0

SERT+/+ SERT+/SERT-/a

**

a

***

Basal

*

Cext Ed (%)

Figure 2

37

Figure 2: (A) Linear regression of zero net flux data from striatum in SERT knockout mice. The x-intercepts estimate extracellular 5-HT levels (Cext) while the slopes of the lines are in vivo extraction fraction (Ed). (B) Mean dialysate 5-HT levels without correction for in vivo extraction fraction (Basal), mean extracellular 5-HT levels (Cext) with correction for in vivo extraction fraction and the corresponding mean in vivo extraction fractions (Ed) in SERT+/+, SERT+/- and SERT-/- mice (n = 10, 9 and 7, respectively).

38

300 200 100 0 -100

SERT+/+ SERT-/-

100

300 [5-HT] in

500

700

Figure 3

Figure 3: High concentrations of Cin (100, 300, 500 and 700 nM) were perfused into striatum of SERT+/+ and SERT-/- mice (n = 7 and 5, respectively) to better determine the slopes of the zero net flux lines or extraction fraction, Ed. This slope is often referred to as "in vivo recovery" and is postulated to be correlated with transporter function.

39

A.

20 15 10 5 0 10 -5 -10 20 30 [5-HT] in 40 50 SERT+/+ SERT+/SERT-/-

B.

60 50 40 30 20 10 0 SERT+/+ SERT+/SERT-/-

** ***

a

**

Basal Cext Ed(%)

Figure 4

40

Figure 4: (A) Linear regression of zero net flux data from frontal cortex in SERT knockout mice. The x-intercepts estimate extracellular 5-HT levels (Cext) while the slopes of the lines are in vivo extraction fraction (Ed). (B) Mean dialysate 5-HT levels without correction for in vivo extraction fraction (Basal), mean extracellular 5-HT levels (Cext) with correction for in vivo extraction fraction and the corresponding mean in vivo extraction fractions (Ed) in SERT+/+, SERT+/- and SERT-/- mice (n = 5, 5 and 5, respectively).

41

1200 1000 800 600 400 200 0

SERT+/+ SERT+/SERT-/-

**

*** DOPAC

5-HIAA

Figure 5

Figure 5: Extracellular levels of 5-HIAA in striatum show a gene dose-dependent decrease in SERT+/+, SERT+/- and SERT-/- mice (n = 12, 20 and 10, respectively). No differences are evident in extracellular DOPAC (n = 6, 14 and 9, respectively).

42

15

1.00 0.75 0.50 0.25 0.00

10

5

0

0

100

200

300

400

500

600

[BAM]

SERT+/+ SERT+/SERT-/0 2 4 6 8 10

1/[Sx10 -3 ]

Figure 6

Figure 6: Oxidation of benzylamine (BAM) by MAO-B to form H2O2 in SERT+/+, SERT+/- and SERT-/- mice. Reduction of H2O2 by horseradish peroxidase coupled to the oxidation of Amplex Red yields the fluorescent product resorufin (RES) in a 1:1 ratio. Mitochondrial preparations from whole brains of the three genotypes of mice were incubated with varying concentrations of benzylamine (10-550 mM). Graph inset shows representative saturation curves from SERT+/+, SERT+/- and SERT-/- mice run on the same day. Points in the Lineweaver-Burke double reciprocal plots are the mean values for each substrate concentration run in triplicate on three separate occasions.

43

1.25

1.00

0.75

1.00

0.50

0.25

0.00

0.75

0

10

20

30

40

50

[Trp]

0.50 SERT+/+ SERT+/SERT-/0.1 0.2

0.25

0.00 0.0

1/[S]

Figure 7

Figure 7: Oxidation of tryptamine (TRP) by MAO-A to form indole-3-acetic acid (I3AA) in the presence of NAD in SERT+/+, SERT+/- and SERT-/- mice. Mitochondrial preparations from whole brains of the three genotypes of mice were incubated with varying concentrations of tryptamine (4-50 mM). Graph inset shows representative saturation curves from SERT+/+, SERT+/- and SERT-/- mice run on the same day. Points in the Lineweaver-Burke double reciprocal plots are the mean values for each substrate concentration run in duplicate on three separate occasions.

44

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