INTRODUCTION

Depression is a common psychological disorder that affects ~7% of the US population (Kessler et al, 2005), with lifetime prevalence approaching 17% (Kessler, 2005). The presynaptic serotonin (5-HT) transporter (SERT) is critical for terminating 5-HT signaling, and changes in SERT expression and/or activity have been proposed to underlie multiple behavioral disorders including depression, obsessive compulsive disorder, and autism (Arango et al, 1995; Malison et al, 1998; Mann et al, 2000; Owens and Nemeroff, 1994; Veenstra-VanderWeele et al, 2012; Ye and Blakely, 2011). Polymorphisms in the SERT promoter region have been reported to influence SERT gene and protein expression (Lesch et al, 1996) and influence sensitivity to life stressors, suggesting a gene-by-environment interaction (Caspi et al, 2003). The 5-HT selective reuptake inhibitors (SSRIs), which antagonize SERT and prolong in vivo 5-HT clearance, are the most common class of antidepressant medications. One challenge to the theory that 5-HT reuptake blockade solely mediates the antidepressant efficacy of SSRIs is that in vivo SERT antagonism occurs within minutes of SSRI administration, whereas therapeutic efficacy requires 4–6 weeks of treatment. Although such a difference can be reconciled through the consideration of time-dependent plasticities arising as a consequence of sustained SERT antagonism, SSRIs have been found to engage other targets that could also contribute to efficacy (Bianchi, 2008; Owens et al, 1997), particularly considering the CNS drug concentration range (1–10 μM) needed for therapeutic effect (Karson et al, 1993; Meyer et al, 2004). These targets include K+, Na+, and Ca++ channels (for review, see Bianchi, 2008), GABA-A (Robinson et al, 2003), nACh (García-Colunga et al, 1997), σ-1 (Villard et al, 2011), and 5-HT receptors (Owens et al, 1997). Elucidating a role for these targets in SSRI action is presumed to inform the development of medications with an improved side-effect profile, though several have been proposed to also contribute to antidepressant action (Heurteaux et al, 2006; Saarelainen et al, 2003; Villard et al, 2011).

SERT knockout mice are an obvious choice as a tool for elucidating SERT-dependent and -independent effects of SSRIs. These mice, however, display major molecular and behavioral compensations, notably displaying spontaneous depression-like phenotypes in various behavioral assays (Lira et al, 2003), reduced CNS 5-HT (Bengel et al, 1998), 5-HT neuron number (Lira et al, 2003), changes in 5-HT receptor expression (Cour et al, 2001; Fabre et al, 2000; Li et al, 2000; Rioux et al, 1999), and abnormal dorsal raphe firing rates (Lira et al, 2003). These aberrations in normal anatomy, physiology, and behavior make it difficult to attribute the effects of SSRIs to a loss of SERT activity per se. In order to better understand the direct and indirect effects of SERT targeted drugs, we sought to create an in vivo model wherein SERT drug recognition is perturbed without compromising SERT surface expression and 5-HT transport. We had observed that human and Drosophila melanogaster SERT (hSERT and dSERT, respectively) display markedly different sensitivities to most SSRIs, though they exhibit equivalent 5-HT recognition. Using cross-species chimera and site-directed mutagenesis methods, we established that high-affinity antagonist recognition depends on a single residue near the 5-HT-binding site (Ile172 in human and mouse, Met167 in fly) (Henry et al, 2006). Site-directed mutagenesis studies revealed that a Met172 substitution in either human or mouse SERT reduces potency for many (but not all, eg, paroxtine) SERT antagonists without affecting the interactions of 5-HT or other substrates (Henry et al, 2006). These in vitro studies encouraged our subsequent creation of the SERT Met172 knock-in mouse (Thompson et al, 2011), whose analysis revealed a loss of SSRI-induced prolongation of 5-HT clearance in vivo, accompanied by a failure to elevate extracellular 5-HT.

Although in the latter studies, we obtained evidence consistent with SERT antagonism as contributing to the acute behavioral actions of SSRIs, the background strain of these mice (129S6/S4) resulted in pro-depressive drug action or inadequate effect sizes to support strong conclusions (Thompson et al, 2011). In order to overcome these issues, and to extend our work to paradigms that better align with the chronic treatments needed for clinical effects, we sought to move the Met172 allele onto a more suitable genetic background. Here we report the results of both acute and chronic SSRI studies utilizing SERT Met172 mice established as congenic on a C57BL/6J background. Our data provide strong evidence that two of the most widely prescribed SSRIs, fluoxetine and citalopram, require SERT blockade to induce behavioral and cellular changes considered predictive of clinical antidepressant efficacy.

MATERIALS AND METHODS

Animals

SERT Met172 knock-in mice on a 129S6/S4 background were created as described previously (Thompson et al, 2011). To move to a background more conducive for the study of SSRI sensitivity (Crowley et al, 2005; Gingrich and Hen, 2000; Jacobson and Cryan, 2007; Lucki et al, 2001), we backcrossed animals until the SERT Met172 allele was >99% congenic onto a C57BL/6J background using a single-nucleotide polymorphism-based speed congenic approach (Jackson Labs, Bar Harbor, Maine, USA). Because the SERT gene (Slc6a4) in the 129S6 embryonic stem cells used to generate the SERT Met172 knock-in line harbors a functional, two amino-acid difference with the C57BL/6J SERT (129: E39R152 haplotype; C57: G39K152 haplotype; Carneiro et al, 2009), it was also necessary to establish a second backcrossed line where the wild-type (WT) 129S6 Slc6a4 gene was expressed on a C57BL/6J background, also established as >99% congenic. A cross of the latter line with the C57BL6/J SERT Met172 mice yields mice heterozygous for either SERT Ile172 or SERT Met172 (SERTI172/M172) with both Slc6a4 alleles sharing the E39R152 haplotype. These heterozygous mice were the parental animals for the homozygous SERT Ile172 (WT) and SERT Met172 mice used in our studies. Initial characterizations of SERT levels, activity, and monoamine homeostasis were performed upon homozygous mice generated from heterozygous breedings. Because SERT levels, 5-HT clearance, raphe 5-HT neuron firing rates, growth rates, and basal tail suspension test (TST) and forced swim test (FST) behaviors were found to be equivalent in heterozygous bred SERT Ile172 and Met172 animals on both the 129S6/S4 (Thompson et al, 2011) and C57BL/6J backgrounds (these studies) we performed chronic drug treatment studies on WT and SERT Met172 mice generated from homozygous breedings. All animal studies were performed in accordance with protocols approved by the Vanderbilt University Animal Care and Use Committee.

Drugs

All general biochemical reagents were obtained from Sigma-Aldrich (St Louis, MO, USA) unless otherwise noted, and were of the highest grade available. Fluoxetine HCl, citalopram HBr, and paroxetine HCl were acquired from TCI Chemicals (Portland, OR, USA). Before i.p. injections and subsequent acute behavioral studies, drugs were prepared fresh in 0.9% sterile saline and filtered for sterility (Nalgene syringe filter, 0.2 μm, 195–2520, ThermoFisher Scientific; Waltham, MA, USA). For chronic administration studies, drugs were dissolved in tap water and filtered for sterility (Stericup-HV, 0.45 μm, PVDF; SCHVU05RE, Millipore, Billerica, MA, USA). Drinking rates were monitored and found to be unaffected between genotypes or by drug treatments (data not shown).

Quantitation of SERT Protein

Following killing by rapid decapitation, brain tissue from 8-week-old male mice was dissected and subjected to western blotting analysis for SERT protein. Samples were resolved on 10% gels by SDS-PAGE, gels were transferred overnight to PVDF membrane (Immobilon, IPVH00010, Millipore), and then SERT was probed using guinea pig anti-SERT primary antibody (Frontier, 5HTT-GP-Af1400, 1 : 1000; Japan; 24 h at 4 °C) and rabbit anti-guinea pig (1 : 10 000; A-5545, Sigma; 1 h at room temperature) as secondary antibody. Bands were detected by enhanced chemiluminescence (Clarity ECL substrate, Bio-Rad, Hercules, CA, USA). Membranes were stripped and loading was normalized after probing for mouse anti-β-actin (Sigma-Aldrich, A5441, 1 : 10 000; 1 h at room temperature) and goat anti-mouse secondary antibody (1 : 10 000; 115-035-062, Jackson ImmunoResearch, West Grove, PA, USA; 1 h at room temperature). Blots were scanned and band density was quantified using ImageJ (http://imagej.nih.gov/ij/).

Brain Monoamine Levels

Male mice (8 weeks) were killed by rapid decapitation and brain regions were dissected on ice and then rapidly frozen on ethanol/dry ice and stored at −80 °C until extraction for assessment of monoamine neurotransmitters and metabolites. Monoamine levels were determined by HPLC/EC methods as described previously (Thompson et al, 2011) in the Vanderbilt Brain Institute Neurochemistry Core. Metabolite levels were normalized to tissue extract protein concentration (Pierce BCA, #23225; ThermoFisher Scientific).

Synaptosomal 5-HT Uptake

Following killing via rapid decapitation, the midbrains of 8–12-week-old male mice were dissected on ice and used for synaptosomal preparations as described previously (Thompson et al, 2011). For competition uptake assays, equal volumes of synaptosomes were incubated with 20 nM [3H]5-HT (PerkinElmer, NET498001MC, Waltham, MA, USA) and varying concentrations of inhibitors and incubated at 37 °C for 10 min. For saturation uptake assays, synaptosomes were prepared using whole brain. Synaptosomes were incubated at 37 °C for 5 min with serial dilutions of 5-HT stock containing 10% [3H]5-HT. At each concentration of 5-HT, parallel samples were incubated in the presence of 1 μM paroxetine, defining non-specific uptake, which was subtracted from total counts to yield specific uptake. Synaptosomes were assessed for protein concentration for the normalization of 5-HT levels across experiments (Pierce BCA; ThermoFisher).

Behavioral Studies

All behavioral assays were performed upon behaviorally naive 8–12-week-old male mice in the Neurobehavioral Core lab at the Vanderbilt University Medical Center. Animals were held at 12-h light:dark cycle (light cycle: 0700 to 1900 hours), and all experimentation occurred between 1300 and 1800 hours. Animals were transferred to housing within the facility and allowed to acclimate for at least 1 week before behavioral manipulations. For acute drug studies, animals were acclimated to handling the day before experimentation, at which point body weight was measured for drug dosing considerations. All drugs for acute drug studies were prepared fresh and administered in 0.9% sterile saline solution at 20 mg/kg (10 μl/g body weight). This dose was utilized to match our prior studies where we demonstrated significant functional SERT occupancy in vivo in WT, but not Met172 mice (Thompson et al, 2011), as assessed by microdialysis studies, and representing a relatively high behaviorally relevant dose (Crowley et al, 2005), which would maximize the potential to detect off-target effects. All experiments and data analyses were scored manually and performed blind to drug condition and genotype.

Tail suspension test

Mice were injected (i.p.) with saline vehicle, fluoxetine, citalopram, or paroxetine 30 min before a 6-min TST. Mice were then suspended by taping the tail to a vertical aluminum bar and activity was recorded by video. Immobility was quantified as the time when mice are motionless, excluding minute limb movements.

Forced swim test

Mice were injected (i.p.) with saline vehicle, fluoxetine, citalopram, or paroxetine 30 min before a 6-min FST. Mice were placed in the center of a 15-cm diameter clear plexiglass cylinder filled with tap water (25–27 °C) to a depth of ~15 cm for the 6 min FST and activity was recorded by video. Immobility was quantified as the time when mice floated or only made movements to maintain balance.

Novelty induced hypophagia

Animals were trained to consume a palatable substance (Vanilla Ensure) in their home cage under low red light conditions (~50 lumens) in the testing room for a total of 3 days of 30-min sessions. On the first day of testing, mice were moved to a novel cage with no bedding and high white light illumination (~1200 lumens), where the latency to first consume Vanilla Ensure, as well as the amount consumed after 30 min (in grams), were measured. On the following day, latency and consumption values of Vanilla Ensure were assessed in the home cage under low light. To avoid ordering effects, the two testing days were switched for half of the mice. We administered citalopram, fluoxetine, or paroxetine in the drinking water (160 mg/l) for 28 days before behavioral screening, which was designed to accomplish a ~20 mg/kg-day chronic dosing regimen (David et al, 2009; Santarelli et al, 2003; Warner-Schmidt et al, 2011). Drug dosage was chosen for continuity with the acute drug sensitivity studies described above, as well as producing drug serum concentrations that are on the high end of clinically relevant serum concentrations (Dulawa and Hen, 2005), maximizing the potential to detect off-target activity. Animals were singly housed during training and testing phases.

Hippocampal Neurogenesis

Proliferation

Following chronic administration of SSRIs and behavioral screening in the NIH test, mice were assayed for levels of hippocampal stem cell proliferation. Mice were administered 5-bromo-2′-deoxyuridine (BrdU; 4 × 75 mg/kg i.p., every 2 h; Sigma-Aldrich) to pulse label newly proliferating S-phase mitotic cells ~24 h following the last behavioral test. Mice continued consumption of water delivered antidepressants up through the BrdU injections. Twenty-four hours following the last injection of BrdU, mice were anesthetized via injection of 100 mg/kg i.p. pentobarbital and transcardially perfused with ice-cold PBS, followed by ice-cold 4% paraformaldehyde. Brains were sectioned (40 μm) via freezing stage sliding microtome (Leica, SM2000R, Buffalo Grove, IL, USA). Every sixth section of the hippocampus (plates 41–61 (Paxinos and Franklin, 2004)) was immunostained for BrdU incorporation (mouse anti-BrdU; 1 : 1000; BD#347580; BD Biosciences; Franklin Lakes, NJ, USA; 48 h at 4 °C) and detected following secondary antibody incubation (biotinylated donkey anti-mouse; 1 : 500; PA1-28627; ThermoFisher; 2 h at room temperature), ABC amplification (VectaStain; Vector Labs; Burlingame, CA, USA; 1 h at room temperature), and diaminobenzidine detection. Bright-field stitched images were captured (Zeiss Axio Imager.M2) and stored for analysis. BrdU+ cells in both sides of the subgranular zone (SGZ) of the dorsal and ventral hippocampus were counted using the ITCN (Image-based Tool for Counting Nuclei) plugin for ImageJ (Byun et al, 2006) by an observer blinded to genotype and drug treatment. Total number of proliferating stem cells was estimated based on counts obtained in every sixth section (yielding, on average, eight sections/mouse) and the total number of sections encompassing the hippocampus.

Survival

We injected a separate cohort of mice not used for behavioral experimentation with BrdU prior to administration of SSRIs. Proliferating cells at steady state were pulse labeled with BrdU (4 × 75 mg/kg i.p., every 2 h), and then their drinking water was supplanted with SSRI-infused drinking water (at 160 mg/l) 24 h after the last BrdU injection. After 4 weeks of SSRI administration, mice were killed and brain tissue collected and developed using the immunohistochemistry procedures described above. BrdU+ cell quantification for proliferation measures included the SGZ and the granule cell layer of the dentate. Cell counting was performed as described above.

Graphical and Statistical Methods

All data were graphed and subjected to statistical analyses using Prism 6.0 (GraphPad, Inc., La Jolla, CA). In all statistical tests, P-value significance thresholds were set to α=0.05. Specific statistical tests to assess main effects and post hoc tests, as well as N for all groups are provided in the figure legends.

RESULTS

SERT Met172 Mice Display Normal SERT Expression, Monoamine Levels, and Saturation Uptake Kinetics

To initiate our studies, we sought to confirm that the SERT Met172 mutation did not alter SERT protein expression, 5-HT recognition, or uptake activity when expressed on a C57BL/6J background. Indeed, quantitative western blot analysis of SERT protein expression revealed no differences in cortical, hippocampal, or midbrain SERT protein levels between WT and SERT Met172 mice (Figure 1a). Similarly, we found no genotype effects on ex vivo 5-HT transport kinetics in whole-brain synaptosomes, neither in 5-HT Km or 5-HT transport Vmax (P>0.05; Figure 1b). Last, monoamine neurotransmitter and metabolite levels did not differ between genotypes (Table 1). Together, these data indicate a lack of consequential effects of the SERT Met172 mutant on basal SERT expression and function, as well as monoamine neurotransmitter homeostasis, aligning with our prior studies (Henry et al, 2006; Thompson et al, 2011).

Figure 1
figure 1

The SERT Met172 mutant imposes altered pharmacological sensitivity with normal SERT expression and function. (a) SERT protein expression was measured via western blotting (right), and normalized to β-actin levels (left). SERT expression did not differ between WT and Met172 mice (two-tailed Student’s t-test, P>0.05; n=5 per genotype). (b) Saturation uptake kinetics in WT and SERT Met172 whole-brain synaptosomes. WT and Met172 do not differ in 5-HT transport activity (WT: Km 73.04±15.66 nM; Vmax 1.18±0.07 pmol/min-mg protein; Met172: Km 102.0±24.61 nM; Vmax 1.22±0.09 pmol/min-mg protein: two-tailed unpaired Student’s t-test, P>0.05, n=6 per genotype). (c) Competition 5-HT uptake analysis. Fluoxetine (WT KI=8.54±0.02 × 10−8 M, SERT Met172 KI=9.03±0.03 × 10−7 M), citalopram (WT KI=2.07±0.05 × 10−8 M, SERT Met172 KI=7.91±0.05 × 10−6 M), and paroxetine (WT KI=2.00±0.03 × 10−9 M, SERT Met172 KI=1.80±0.04 × 10−9 M) were assessed for their ability to compete with [3H]5-HT uptake. (n=4 per genotype and condition.)

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Table 1 Values Presented as Mean±SEM (ng/mg Protein)
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SERT Met172-Derived Synaptosomes Display Reduced Sensitivity to SERT Antagonists

After confirming normal SERT expression and activity, we sought to verify whether the C57BL/6J backcrossed SERT Met172 allele maintained its reduced sensitivity to specific SERT antagonists (Henry et al, 2006; Thompson et al, 2011). To accomplish this, we performed competitive [3H]5-HT uptake using midbrain-derived synaptosomes of WT and SERT Met172 mice (Figure 1c). These studies revealed the expected reductions in potency for fluoxetine (~20 fold) and citalopram (~500 fold) at SERT Met172. In contrast, paroxetine was unaffected by the Met172 mutant, as demonstrated by its equivalent potency of 5-HT uptake inhibition in WT, consistent with predictions from transfected cell studies (Henry et al, 2006).

SERT Met172 Mice are Insensitive to Acute Fluoxetine and Citalopram in the TST and FST

Having validated the C57BL/6J SERT Met172 model as having normal SERT expression and function, yet disrupted ex vivo sensitivity to the SSRIs fluoxetine and citalopram, we moved to assess the efficacy of these drugs in behavioral assays that are known to be sensitive to acute antidepressant administration (Porsolt et al, 1977; Steru et al, 1985). Clinical antidepressants—including fluoxetine, citalopram, and paroxetine—are known to promote escape-like behavior in mice when presented with an inescapable stressor, such as suspension by the tail (TST) or placement in a cylinder of water (FST). As expected, WT mice showed a robust decrease in immobility following acute fluoxetine and citalopram administration (Figure 2a and b), with effect sizes that match those observed in the literature accounting for age, genetic background strain, and drugs utilized (Crowley et al, 2005). SERT Met172 mice, however, displayed no response to either drug (Figure 2a and b). Importantly, paroxetine equivalently suppressed immobility in WT and SERT Met172 mice in both the TST and FST, consistent with the inability of the Met172 substitution to perturb paroxetine interactions. This important control also demonstrates a normal capacity of the SERT Met172 mice to translate SERT inhibition and elevations in extracellular 5-HT into enhanced mobility in both tests.

Figure 2
figure 2

Actions of fluoxetine and citalopram in the TST and FST arise from SERT antagonism. All tests were performed 30 min after i.p. injection of 20 mg/kg drug. (a) Time immobile in a 6-min TST. Two way ANOVA revealed significant main effects of genotype (F(1,72)=17.11, P<0.05), treatment (F(3,72)=10.82, P<0.05), and interaction effect (F(3,72)=4.62, P<0.05). WT mice display significant decreases in immobility time in response to all drug treatments. Met172 mice display significant decreases in immobility time only in response to paroxetine. (b) Time immobile in a 6-min FST. Two way ANOVA revealed significant effects of genotype (F(1,71)=18.80, P<0.05), treatment (F(3,71)=15.92, P<0.05), and interaction effect (F(3,71)=4.52, P<0.05). WT mice display significant decreases in immobility time in response to all drug treatments. Met172 mice display significant decreases in immobility time only in response to paroxetine. For a–b, * indicates significance (P<0.05) compared with vehicle treatment via Bonferonni post hoc tests (n=10–12 per genotype and treatment condition).

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SERT Met172 Mice Fail to Respond to Chronic Fluoxetine and Citalopram in the NIH Test

Although the FST and TST are tests with predictive validity for antidepressant efficacy, we sought to evaluate the SERT dependency of SERT actions in the Met172 model using tests that more closely mirror the time course associated with SSRI clinical efficacy. Thus, we implemented the NIH test, a behavioral paradigm sensitive to chronic, but not acute, antidepressant administration (Dulawa and Hen, 2005). The NIH test involves monitoring the latency of animals to approach and consume a known palatable substance in a novel, stressful environment, where SSRIs reduce latency and enhance consumption after chronic but not acute administration (Dulawa and Hen, 2005). We administered citalopram, fluoxetine, or paroxetine in the drinking water (160 mg/l) for 28 days before behavioral screening. Following SSRI administration, WT mice displayed a reduced latency to consume Vanilla Ensure in the novel cage compared with vehicle condition (Figure 3a), with effect sizes comparable to those reported by others (Dulawa and Hen, 2005; Santarelli et al, 2003). SERT Met172 mice administered paroxetine displayed similar reductions in latency as WT animals, whereas those provided fluoxetine or citalopram showed no significant reductions relative to vehicle condition. Paralleling our findings with latency, WT mice also displayed increased novel cage consumption following all SSRI treatments, whereas SERT Met172 mice displayed increased consumption only with paroxetine administration (Figure 3b).

Figure 3
figure 3

SERT antagonism is required for the chronic effects of fluoxetine and citalopram in the NIH test. (a) Latency to consume Ensure in novel cage was recorded. Two way ANOVA revealed significant main effects of genotype (F(1,97)=4.81, P<0.05) and treatment (F(3,97)=5.97, P<0.05), but not an interaction effect (F(3,97)=1.92, P>0.05). WT mice display significant reductions in latency following chronic SSRI administration, whereas Met172 mice display significant reductions only following paroxetine administration. (b) Consumption effects: mice were left in novel cage for a total of 30 min and allowed to freely consume vanilla Ensure. Two way ANOVA revealed significant main effects of genotype (F(1,97)=8.53, P<0.05) and treatment (F(3,97)=6.10, P<0.05), but not an interaction effect (F(3,97)=1.46, P>0.05). WT mice significantly increase consumption following chronic SSRI administration. SERT Met172 mice only increase consumption following paroxetine administration. For a–b, * indicates significance (P<0.05) compared with vehicle treatment Bonferroni post hoc tests (n=12–18 per genotype and treatment condition).

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The Ability of Chronic Fluoxetine and Citalopram to Promote SGZ Stem Cell Proliferation and Survival is Dependent upon SERT Antagonism

Hippocampal neurogenesis is a non-behavioral measure of antidepressant efficacy that has been shown to be sensitive to chronic, but not acute, antidepressant administration, and irradiation studies demonstrate neurogenesis as essential for SSRI efficacy in the NIH test (Santarelli et al, 2003). SSRIs promote both hippocampal stem cell proliferation rate and survival of newly generated hippocampal stem cells (Wang et al, 2008). Consistent with these studies, chronic administration of fluoxetine, citalopram, and paroxetine robustly stimulated the proliferation rate (Figure 4a and b) and survival (Figure 4c) of hippocampal stem cells in WT mice, assessed via BrdU+ immunohistochemistry. The magnitude of these elevations in hippocampal neurogenesis measures parallel previous observations (Santarelli et al, 2003). In contrast, only paroxetine was able to enhance hippocampal stem cell proliferation (Figure 4a and b) or survival (Figure 4c) in SERT Met172 mice.

Figure 4
figure 4

SERT antagonism is required for the stimulation of hippocampal stem cell proliferation and survival following chronic fluoxetine and citalopram. (a) Example images of hippocampal stem cell proliferation studies. Scale bar represents 100 μm. Dotted line represents the border of the of the granular cell layer of the dentate gyrus for visual aid. (b) Proliferation: following administration of SSRIs, WT mice display significant increases in stem cell proliferation rate. Met172 mice display significant increases in proliferation only after paroxetine. Two way ANOVA revealed significant effects of genotype (F(1,19)=4.60, P<0.05), treatment (3,19)=8.88, P<0.05), and interaction effect (F(3,19)=5.06, P<0.05). (c) Survival: newly generated stem cells survive significantly more in SSRI-treated mice than vehicle. SERT Met172 mice display increased survival rates only after paroxetine. Two way ANOVA revealed significant main effect of genotype (F(1,22)=6.04, P<0.05) and treatment (F(3,22)=6.03, P<0.05), but not on interaction effect (F(3,22)=2.47, P>0.05. For b–c, * indicates significance compared with vehicle (P<0.05) following Bonferroni post hoc tests (n=4–5 per genotype and condition).

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DISCUSSION

The development of SSRIs arose from basic and clinical research that supported a role for 5-HT signaling alterations in depression, and from observations that drugs that increased 5-HT signaling could alleviate depressive symptoms (López-Muñoz and Alamo, 2009). Focusing upon SERT inhibition as a powerful means of 5-HT signal enhancement, multiple SSRIs gained regulatory approval for the treatment of depression and related mood disorders. Although these agents were designed to be SERT specific, a limited capacity to assess the full ‘druggable genome’ has precluded exclusion of major or minor contributions from other targets. Indeed, multiple proteins have been suggested to interact with various antidepressants with sufficient affinity to warrant further consideration as contributing to antidepressant efficacy (Heurteaux et al, 2006; Saarelainen et al, 2003). In addition, the significant incongruity between the timescale of in vivo SERT antagonism and clinical benefit has raised concerns that other targets may participate, though time-dependent plasticity downstream of 5-HT receptors is a commonly invoked explanation. Indirect support for SERT’s primacy in antidepressant action is provided by the existence of multiple, structurally diverse SSRIs that share SERT antagonism but (presumably) differ in off-target actions, as well as the observations that certain 5-HT receptor antagonists can attenuate the effects of SSRIs in preclinical models (Cryan and Lucki, 2000).

Efforts to use SERT KO mice to validate an essential role of SERT blockade in SSRI actions (Holmes et al, 2002) are confounded by the significant compensatory alterations that arise in the model. With these issues in mind, we developed the SERT Met172 mouse model as a potentially powerful tool for extracting the SERT contribution from both SERT-selective and more broadly acting drugs. Our data demonstrate a requirement for SERT antagonism, and by inference, elevations in extracellular 5-HT and 5-HT receptor activation, in both the acute and chronic behavioral actions of the SSRIs fluoxetine and citalopram. We also established that the effects of these drugs in promoting hippocampal stem cell expansion and survival require SERT blockade. Prior in vitro studies with antidepressants found effects on cultured stem cells that lack a serotonergic innervation (Chang et al, 2010), suggesting that SSRIs might have cell autonomous actions on stem cell proliferation and survival. Though our studies do not rule out an auxiliary activity of one or more SSRIs on stem cell proliferation or survival, they indicate that, in vivo, potentiated 5-HT signaling is essential to the well-replicated ability of SSRIs to stimulate subgranular stem cell expansion.

The studies presented in our prior and current reports amply demonstrate the utility of the SERT Met172 model for defining actions of SSRIs as SERT dependent or independent. SSRIs exhibit a number of actions that limit therapeutic benefit, such as effects on libido, weight gain, sleep, and gastrointestinal function that for some individuals can prove unacceptable. Tests that evaluate such actions should benefit from inclusion of the SERT Met172 model to probe their reliance on augmented 5-HT signaling. As observed with paroxetine, however, not all SERT blockers are sensitive to the Met172 substitution, and thus in vitro studies for inhibition of [3H]5-HT uptake are utilized to assess pharmacological sensitivity before further in vivo assessments. With respect to the utility of the SERT Met172 model in extracting the contribution of 5-HT signaling to mixed-action drugs, we recently capitalized on the model to demonstrate an essential role of SERT blockade in the ability of cocaine to shift circadian rhythms in neuronal firing and locomotor behavior (Prosser et al, 2014). In addition, multiple medications exist that combine SERT inhibition with actions at other targets, including duloxetine, venlafaxine, vortioxetine, and milnacipran. The SERT Met172 model could be helpful in preclinical efforts to determine the degree to which SERT antagonism contributes to the therapeutic efficacy of these agents. Finally, the SERT Met172 model should be of value in elucidating the 5-HT origins of the constellation of molecular plasticities that arise with chronically administered drugs sensitive to the substitution. Using various ‘omic’ strategies, we envisage exploiting the SERT Met172 model to tag molecular changes as SERT dependent or independent, information that can help refine the definition of mechanisms impacting efficacy and treatment onset.

Funding and Disclosure

Research support was provided by the National Institutes of Health through awards MH094527 (RDB) and MH096972 (RDB and JVV). The Vanderbilt Kennedy Center for Research on Human Development (P30 HD15052) provided infrastructure support for behavioral and neurochemical analyses. Dr Blakely has consulted with Forest, Lundbeck, Wyeth, Pfizer, and Prexa and has received research funding from Forest, Lundbeck, Wyeth, and Prexa. Dr Veenstra-VanderWeele has consulted with Roche, Novartis, and Synapdx, and has received research funding from Roche, Novartis, SynapDx, Seaside Therapeutics, and Forest.