Meredith A. Fox

How the Serotonin Story is Being Rewritten By New Gene-Based Discoveries Principally Related to SLC6A4, the Serotonin Transporter Gene, Which Functions To Influence All Cellular Serotonin Systems

Discovered and crystallized over sixty years ago, serotonins important functions in the brain and body were identified over the ensuing years by neurochemical, physiological and pharmacological investigations. This 2008 M. Rapport Memorial Serotonin Review focuses on some of the most recent discoveries involving serotonin that are based on genetic methodologies. These include examples of the consequences that result from direct serotonergic gene manipulation (gene deletion or overexpression) in mice and other species; an evaluation of some phenotypes related to functional human serotonergic gene variants, particularly in SLC6A4, the serotonin transporter gene; and finally, a consideration of the pharmacogenomics of serotonergic drugs with respect to both their therapeutic actions and side effects. The serotonin transporter (SERT) has been the most comprehensively studied of the serotonin system molecular components, and will be the primary focus of this review. We provide in-depth examples of gene-based discoveries primarily related to SLC6A4 that have clarified serotonins many important homeostatic functions in humans, non-human primates, mice and other species.

Hypolocomotion, anxiety and serotonin syndrome‐like behavior contribute to the complex phenotype of serotonin transporter knockout mice

Although mice with a targeted disruption of the serotonin transporter (SERT) have been studied extensively using various tests, their complex behavioral phenotype is not yet fully understood. Here we assess in detail the behavior of adult female SERT wild type (+/+), heterozygous (+/−) and knockout (−/−) mice on an isogenic C57BL/6J background subjected to a battery of behavioral paradigms. Overall, there were no differences in the ability to find food or a novel object, nest‐building, self‐grooming and its sequencing, and horizontal rod balancing, indicating unimpaired sensory functions, motor co‐ordination and behavioral sequencing. In contrast, there were striking reductions in exploration and activity in novelty‐based tests (novel object, sticky label and open field tests), accompanied by pronounced thigmotaxis, suggesting that combined hypolocomotion and anxiety (rather than purely anxiety) influence the SERT −/− behavioral phenotype. Social interaction behaviors were also markedly reduced. In addition, SERT −/− mice tended to move close to the ground, frequently displayed spontaneous Straub tail, tics, tremor and backward gait – a phenotype generally consistent with ‘serotonin syndrome’‐like behavior. In line with replicated evidence of much enhanced serotonin availability in SERT −/− mice, this serotonin syndrome‐like state may represent a third factor contributing to their behavioral profile. An understanding of the emerging complexity of SERT −/− mouse behavior is crucial for a detailed dissection of their phenotype and for developing further neurobehavioral models using these mice.

A pharmacological analysis of mice with a targeted disruption of the serotonin transporter

RationalePartial or complete ablation of serotonin transporter (SERT) expression in mice leads to altered responses to serotonin receptor agonists and other classes of drugs.ObjectivesIn the current report, we review and integrate many of the major behavioral, physiological, and neurochemical findings in the current literature regarding pharmacological assessments made in SERT mutant mice.ResultsThe absence of normal responses to serotonin reuptake inhibiting (SRI) antidepressants in SERT knockout (−/−) mice demonstrates that actions on SERT are a critical principle mechanism of action of members of this class of antidepressants. Drugs transported by SERT, (+)-3,4-methylenedioxymethamphetamine (MDMA) and 1-methyl-4-(2′-aminophenyl)-1,2,3,6-tetrahydropyridine (2′-NH2-MPTP), are also inactive in SERT −/− mice. Temperature, locomotor, and electrophysiological responses to various serotonin receptor agonists, including 8-hydroxy-2-(di-n-propylamino)-tetraline (8-OH-DPAT), ipsapirone, and RU24969, are reduced in SERT −/− mice, despite comparatively lesser reductions in Htr1a and Htr1b binding sites, G-proteins, and other signaling molecules. SERT −/− mice exhibit an ∼90% reduction in head twitches in response to the Htr2a/2c agonist (+/−)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), associated with a profound reduction in arachidonic acid signaling, yet only modest changes in Htr2a and Htr2c binding sites. SERT −/− mice also exhibit altered behavioral responses to cocaine and ethanol, related to abnormal serotonin, and possibly dopamine and norepinephrine, homeostasis.ConclusionsTogether, these studies demonstrate a complex and varied array of modified drug responses after constitutive deletion of SERT and provide insight into the role of serotonin, and in particular, its transporter, in the modulation of complex behavior and in the pharmacological actions of therapeutic agents and drugs of abuse.

Receptor mediation of exaggerated responses to serotonin-enhancing drugs in serotonin transporter (SERT)-deficient mice

Administration of serotonin-enhancing drugs induces a distinctive behavioral syndrome in rodents. We previously reported that mice with a targeted disruption of the serotonin transporter (SERT) display some of these behaviors spontaneously, in the absence of drug. In the current studies, we assessed the drug-induced serotonin syndrome in SERT wildtype (+/+), heterozygous (+/-) and knockout (-/-) mice. In SERT -/- mice, the monoamine oxidase inhibitor (MAOI) tranylcypromine (1mg/kg) or the serotonin precursor 5-hydroxy-L-tryptophan (5-HTP; 80 mg/kg) led to markedly exaggerated serotonin syndrome behaviors relative to SERT +/+ mice, with an intermediate phenotype in SERT +/- mice. SERT +/+ mice developed significant serotonin syndrome behaviors only with the combination of the MAO-A/B inhibitor tranylcypromine (0.5 or 1 mg/kg) or the MAO-A-selective inhibitor clorgyline (1.2 mg/kg) plus 5-HTP. In evaluations of underlying mechanisms, pretreatment with the Htr1a receptor antagonist WAY 100635 (1 mg/kg), but not the Htr7 antagonist SB 269970 (3 mg/kg) or the Htr2a antagonist MDL 11,939 (5 mg/kg), markedly decreased the exaggerated 5-HTP-induced behaviors in SERT -/- mice. Subsequent experiments showed that the Htr1a agonist 8-OH-DPAT (1 or 2 mg/kg) elicited serotonin syndrome behaviors in a dose-dependent manner, blocked by WAY 100635 (1 mg/kg), in mice of all three genotypes, confirming the role of Htr1a receptors. The current data document markedly enhanced behavioral sensitivity to serotonin-enhancing drugs in SERT-deficient mice. These studies also show that the exaggerated behavioral responses observed in SERT +/- and -/- mice are mediated by postsynaptic Htr1a receptors, and suggest intact postsynaptic Htr1a function in SERT -/- mice.

Neurochemical, behavioral, and physiological effects of pharmacologically enhanced serotonin levels in serotonin transporter (SERT)-deficient mice

RationaleSerotonin transporter (SERT) knockout (−/−) mice have an altered phenotype in adulthood, including high baseline anxiety and depressive-like behaviors, associated with increased baseline extracellular serotonin levels throughout life.ObjectivesTo examine the effects of increases in serotonin following the administration of the serotonin precursor 5-hydroxy-l-tryptophan (5-HTP) in SERT wild-type (+/+), heterozygous (+/−), and −/− mice.Results5-HTP increased serotonin in all five brain areas examined with approximately 2- to 5-fold increases in SERT+/+ and +/− mice, and with greater 4.5- to 11.7-fold increases in SERT−/− mice. Behaviorally, 5-HTP induced exaggerated serotonin syndrome behaviors in SERT−/−, mice with similar effects in male and female mice. Studies suggest promiscuous serotonin uptake by the dopamine transporter (DAT) in SERT−/− mice, and here, the DAT blocker GBR 12909 enhanced 5-HTP-induced behaviors in SERT−/− mice. Physiologically, 5-HTP induced exaggerated temperature effects in SERT-deficient mice. The 5-HT1A antagonist WAY 100635 decreased 5-HTP-induced hypothermia in SERT+/+ and +/− mice with no effect in SERT−/− mice, whereas the 5-HT7 antagonist SB 269970 decreased this exaggerated response in SERT−/− mice only. WAY 100635 and SB 269970 together completely blocked 5-HTP-induced hypothermia in SERT+/− and −/− mice.ConclusionsThese studies demonstrate that SERT−/− mice have exaggerated neurochemical, behavioral, and physiological responses to further increases in serotonin, and provide the first evidence of intact 5-HT7 receptor function in SERT−/− mice, with interesting interactions between 5-HT1A and 5-HT7 receptors. As roles for 5-HT7 receptors in anxiety and depression were recently established, the current findings have implications for understanding the high anxiety and depressive-like phenotype of SERT-deficient mice.

Anxiety and affective disorder comorbidity related to serotonin and other neurotransmitter systems: obsessive–compulsive disorder as an example of overlapping clinical and genetic heterogeneity

Individuals with obsessive–compulsive disorder (OCD) have also been shown to have comorbid lifetime diagnoses of major depressive disorder (MDD; rates greater than 70%), bipolar disorder (rates greater than 10%) and other anxiety disorders (e.g. panic disorder, post-traumatic stress disorder (PTSD)). In addition, overlap exists in some common genetic variants (e.g. the serotonin transporter gene (SLC6A4), the brain-derived neurotrophic factor (BDNF) gene), and rare variants in genes/chromosomal abnormalities (e.g. the 22q11 microdeletion syndrome) found across the affective/anxiety disorder spectrums. OCD has been proposed as a possible independent entity for DSM-5, but by others thought best retained as an anxiety disorder subtype (its current designation in DSM-IV), and yet by others considered best in the affective disorder spectrum. This review focuses on OCD, a well-studied but still puzzling heterogeneous disorder, regarding alterations in serotonergic, dopaminergic and glutamatergic neurotransmission in addition to other systems involved, and how related genes may be involved in the comorbidity of anxiety and affective disorders. OCD resembles disorders such as depression, in which gene × gene interactions, gene × environment interactions and stress elements coalesce to yield OC symptoms and, in some individuals, full-blown OCD with multiple comorbid disorders.

Animal models of the serotonin syndrome: a systematic review.

The serotonin syndrome (SS) is a potentially life-threatening disorder in humans which is induced by ingestion of an overdose or by combination of two or more serotonin (5-HT)-enhancing drugs. In animals, acute administration of direct and indirect 5-HT agonists also leads to a set of behavioral and autonomic responses. In the current review, we provide an overview of the existing versions of the animal model of the SS. With a focus on studies in rats and mice, we analyze the frequency of behavioral and autonomic responses following administration of 5-HT-enhancing drugs and direct 5-HT agonists administered alone or in combination, and we briefly discuss the receptor mediation of these responses. Considering species differences, we identify a distinct set of behavioral and autonomic responses that are consistently observed following administration of direct and indirect 5-HT agonists. Finally, we discuss the importance of a standardized assessment of SS responses in rodents and the utility of animal models of the SS in translational studies, and provide suggestions for future research.

Altered 5-HT2C receptor agonist-induced responses and 5-HT2C receptor RNA editing in the amygdala of serotonin transporter knockout mice

BackgroundThe serotonin 5-HT2C receptor (5-HT2CR) is expressed in amygdala, a region involved in anxiety and fear responses and implicated in the pathogenesis of several psychiatric disorders such as acute anxiety and post traumatic stress disorder. In humans and in rodent models, there is evidence of both anxiogenic and anxiolytic actions of 5-HT2C ligands. In this study, we determined the responsiveness of 5-HT2CR in serotonin transporter (SERT) knockout (-/-) mice, a model characterized by increased anxiety-like and stress-responsive behaviors.ResultsIn the three-chamber social interaction test, the 5-HT2B/2C agonist mCPP decreased sociability and sniffing in SERT wildtype (+/+) mice, both indicative of the well-documented anxiogenic effect of mCPP. This 5-HT2C-mediated response was absent in SERT -/- mice. Likewise, in the open field test, the selective 5-HT2C agonist RO 60-0175 induced an anxiogenic response in SERT +/+ mice, but not in SERT -/- mice. Since 5-HT2CR pre-mRNA is adenosine-to-inosine (A-to-I) edited, we also evaluated the 5-HT2CR RNA editing profiles of SERT +/+ and SERT -/- mice in amygdala. Compared to SERT +/+ mice, SERT-/- mice showed a decrease in less edited, highly functional 5-HT2C isoforms, and an increase in more edited isoforms with reduced signaling efficiency.ConclusionsThese results indicate that the 5-HT2CR in the amygdala of SERT -/- mice has increased RNA editing, which could explain, at least in part, the decreased behavioral responses to 5-HT2C agonists in SERT -/- mice. These alterations in 5-HT2CR in amygdala may be relevant to humans with SERT polymorphisms that alter SERT expression, function, and emotional behaviors.

Functional interactions between 5‐HT2A and presynaptic 5‐HT1A receptor‐based responses in mice genetically deficient in the serotonin 5‐HT transporter (SERT)

Background and purpose:  Despite decreased presynaptic 5‐HT1A and altered 5‐HT2A receptor function in genetically‐deficient serotonin (5‐HT) transporter (SERT) mice, the 5‐HT1A receptor antagonist N‐[2‐[4‐(2‐methoxyphenyl)‐1‐piperazinyl]ethyl]‐N‐2‐pyridinylcyclohexanecarboxamide maleate salt (WAY 100635) still induced head twitches in these mice, a well‐established 5‐HT2A receptor‐mediated response.

Imaging Elevated Brain Arachidonic Acid Signaling in Unanesthetized Serotonin Transporter (5-HTT)-Deficient Mice

Certain polymorphisms reduce serotonin (5-HT) reuptake transporter (5-HTT) function and increase susceptibility to psychiatric disorders. Heterozygous (5-HTT+/−)-deficient mice, models for humans with these polymorphisms, have elevated brain 5-HT concentrations and behavioral abnormalities. As postsynaptic 5-HT2A/2C receptors are coupled to cytosolic phospholipase A2 (cPLA2), which releases arachidonic acid (AA) from membrane phospholipid, 5-HTT-deficient mice may have altered brain AA signaling and metabolism. To test this hypothesis, signaling was imaged as an AA incorporation coefficient k* in unanesthetized homozygous knockout (5-HTT−/−), 5-HTT+/− and wild-type (5-HTT+/+), mice following saline (baseline) or 1.5 mg/kg s.c. DOI, a partial 5-HT2A/2C receptor agonist. Enzyme activities, metabolite concentrations, and head-twitch responses to DOI were also measured. Baseline k* was widely elevated by 20–70% in brains of 5-HTT+/− and 5-HTT−/− compared to 5-HTT+/+ mice. DOI increased k* in 5-HTT+/+ mice, but decreased k* in 5-HTT-deficient mice. Brain cPLA2 activity was elevated in 5-HTT-deficient mice; cyclooxygenase activity and prostaglandin E2 and F2α and thromboxane B2 concentrations were reduced. Head-twitch responses to DOI, although robust in 5-HTT+/+ and 5-HTT+/− mice, were markedly fewer in 5-HTT−/− mice. Pretreatment with para-chlorophenylalanine, a 5-HT synthesis inhibitor, restored head twitches in 5-HTT−/− mice to levels in 5-HTT+/+ mice. We propose that increased baseline values of k* in 5-HTT-deficient mice reflect tonic cPLA2 stimulation through 5-HT2A/2C receptors occupied by excess 5-HT, and that reduced k* and head-twitch responses to DOI reflected displacement of receptor-bound 5-HT by DOI with a lower affinity. Increased baseline AA signaling in humans having polymorphisms with reduced 5-HTT function might be identified using positron emission tomography.