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Objective: Association between the low-activity variant of a polymorphism in the transcriptional control region of the serotonin transporter (5-HTTLPR) and neuroticism or harm avoidance was found in several but not all studies. The authors analyzed the influence of 5-HTTLPR variants on personality disorders., Method: Patients with personality disorders (N=320) and healthy volunteers (N=281) were studied with the Revised NEO Personality Inventory and the Tridimensional Personality Questionnaire. All were genotyped for 5-HTTLPR variants., Results: No differences in 5-HTTLPR genotype distribution were detected between patients with cluster B and C personality disorders and comparison subjects. In contrast, among patients with a cluster C diagnosis, carriers of the low-activity short allele of the 5-HTTLPR exhibited higher neuroticism scores than noncarriers., Conclusions: These findings support the notion that there is no general association between the 5-HTTLPR and anxiety-related traits and that differential gene effects and/or gene-by-environment interactions are likely operative in distinct clinical subpopulations.

Current evidence indicates that virtually all neuropsychiatric disorders, like many other common medical disorders, are genetically complex, with combined influences from multiple interacting genes, as well as from the environment. However, additive or epistatic gene interactions have proved quite difficult to detect and evaluate in human studies. Mouse phenotypes, including behaviors and drug responses, can provide relevant models for human disorders. Studies of gene-gene interactions in mice could thus help efforts to understand the molecular genetic bases of complex human disorders. The serotonin transporter (SERT, 5-HTT, SLC6A4) provides a relevant model for studying such interactions for several reasons: human variants in SERT have been associated with several neuropsychiatric and other medical disorders and quantitative traits; SERT blockers are effective treatments for a number of neuropsychiatric disorders; there is a good initial understanding of the phenotypic features of heterozygous and homozygous SERT knockout mice; and there is an expanding understanding of the interactions between variations in SERT expression and variations in the expression of a number of other genes of interest for neuropsychiatry and neuropharmacology. This paper provides examples of experimentally-obtained interactions between quantitative variations in SERT gene expression and variations in the expression of five other mouse genes: DAT, NET, MAOA, 5-HT(1B) and BDNF. In humans, all six of these genes possess polymorphisms that have been independently investigated as candidates for neuropsychiatric and other disorders in a total of > 500 reports. In the experimental studies in mice reviewed here, gene-gene interactions resulted in either synergistic, antagonistic (including 'rescue' or 'complementation') or more complex, quantitative alterations. These were identified in comparisons of the behavioral, physiological and neurochemical phenotypes of wildtype mice vs. mice with single allele or single gene targeted disruptions and mice with partial or complete disruptions of multiple genes. Several of the descriptive phenotypes could be best understood on the basis of intermediate, quantitative alterations such as brain serotonin differences. We discuss the ways in which these interactions could provide models for studies of gene-gene interactions in complex human neuropsychiatric and other disorders to which SERT may contribute, including developmental disorders, obesity, polysubstance abuse and others.

Variation in the serotonin transporter gene-linked polymorphic region (5-HTTLPR) has been associated with anxiety and harm avoidance and is weakly associated with a number of neuropsychiatric disorders, including Type II alcoholism, which has a high rate of comorbidity with antisocial personality disorder. Studies have also demonstrated interactions between 5-HTLPR variation and environmental stress on the incidence of depression. As in humans, there is a serotonin transporter gene promoter length polymorphism in rhesus macaques that produces similar decreases in transcriptional efficiency. Macaques with histories of early-life stress have been shown to exhibit impulsive aggression, incompetent social behavior and increased behavioral and endocrine responsivity to stress. In this paper, we review studies performed previously in our lab and present preliminary data examining interactions between early rearing and serotonin transporter gene promoter variation on the incidences of play behavior and aggression in infant rhesus macaques. The data presented here highlight the importance of considering gene-environment interactions when studying childhood risk factors for aggression, anxiety and related neuropsychiatric disorders and support the use of the nonhuman primate for studing gene by environment interactions in behavioral research.

Mood, emotion and cognition are modulated by serotonergic neurotransmission, while the physiological function of serotonergic synapses depends on serotonin reuptake, which is mediated by the serotonin transporter (5-HTT). Allelic variation of 5-HTT expression in humans is caused by a functional gene-promoter polymorphism with two predominant variant alleles, which are associated with variations in anxiety measures as previously reported. Here we report that administration of dexamethasone, a potent glucocorticosteroid hormone, results in an increase in 5-HTT expression in immortalized human B-lymphoblastoid cells, which express the human 5-HTT. Functional reporter gene assays as well as 5-HT uptake and inhibitor binding measures revealed a genotype-dependent dose-response to glucocorticosteroid administration, which was antagonized by RU 38486, a non-specific glucocorticosteroid hormone antagonist. The allele-specific differences after administration of dexamethasone depended on the repetitive GC-rich sequence located approximately 1.4 kb upstream of the 5-HTT gene transcription site because of absence of a significant steroid effect after transfecting a deletional mutant reporter gene construct, which lacks this repetitive promoter sequence. Our findings may contribute to explain the vulnerability to stress-related disorders in susceptible individuals, in whom further clinical studies should follow up on these in vitro findings.

The intensity dependence of the auditory evoked potential (AEP) has been suggested as an indicator of central serotonergic function, a strong intensity dependence presumably reflecting low serotonergic activity. As individual differences in serotonergic neurotransmission can be accounted for in part by genetic variation in genes of the serotonergic pathway, we investigated whether a functional polymorphism in the promoter region of the serotonin transporter gene (5-HTTLPR) is associated with the AEP intensity dependence. Because dopaminergic influences on the intensity dependence have also been reported, we furthermore explored the role of a functional polymorphism in the dopamine D4 receptor gene (DRD4 exon III) in the modulation of the AEP intensity dependence. AEPs to tones of six intensity levels were recorded from 60 healthy young individuals, and N1/P2 linear as well as median slopes at central electrode sites were computed as measures of the AEP intensity dependence. Analyses of variance showed that there was a significant effect of the 5-HTTLPR on the AEP intensity dependence. Individuals with the ll genotype exhibited a stronger intensity dependence compared to individuals with the ls genotype. This effect was even more pronounced when DRD4 exon III was considered in the analyses. In conclusion, these findings provide further evidence for a role of serotonergic neurotransmission in the modulation of the AEP intensity dependence. The results also point to possible dopaminergic influences on the AEP intensity dependence., (Copyright 2003 Wiley-Liss, Inc.)

The serotonin transporter (5HTT) is the molecule responsible for the high-affinity reuptake of 5HT from the synaptic cleft. Mice lacking the 5HTT exhibit highly elevated extracellular concentrations of 5HT. We assessed whether the glutathione detoxification system is altered in 5HTT-deficient mice. While levels of reduced and oxidized glutathione were unchanged, glutathione metabolising enzymes showed a differential pattern of modulation. Glutathione peroxidase was reduced in frontal cortex, brainstem, and cerebellum of 5HTT-deficient mice, though not to a statistically significant extent, while a putative isoform of the detoxifying enzyme glutathione-S-transferase pi was decreased in a number of brain regions, especially in brainstem. At the level of the DNA, we found an increase of oxidative DNA adducts in the hippocampus of 5HTT-deficient mice. Given the importance of the hippocampus in learning and memory, this may be the most important neurochemical consequence of the absence of the 5HTT.

Nonhuman primates offer unique opportunities to study the effects of genes, environments, and their interaction, on physiology and complex behavior. We examined genotype and early environment contributions to CNS function in a large sample of rhesus monkeys. In humans, length variation of the serotonin (5-HT) transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) that results in allelic variation in 5-HTT expression is associated with decreased serotonergic function and 5-HT-mediated psychopathology. We report that an analogous variation of the gene's regulatory region in monkeys interacts with early experience to affect central 5-HT functioning. Monkeys with deleterious early rearing experiences were differentiated by genotype in cerebrospinal fluid concentrations of the 5-HT metabolite, 5-hydroxyindoleacetic acid, while monkeys reared normally were not. These findings demonstrate an environment-dependent effect of the rh5-HTTLPR genotype on CNS 5-HT function and suggest nonhuman primates may provide an important avenue for investigating gene/environment interactions using candidate genes for physiological and behavioral traits.

A polymorphism in the serotonin (5-HT) transporter gene regulatory region (5-HTTLPR) is associated with measures of 5-HT transporter (5-HTT) expression and 5-HT-mediated behaviors in humans. An analogous length variation of the 5-HTTLPR has been reported in rhesus monkeys (rh5-HTTLPR). A retrospective association study was conducted on 115 rhesus macaque infants either homozygous for the long 5HTTLPR variant (l/l) or heterozygous for the short and long form (l/s). To assess contributions of genotype and early rearing environment, 36 mother-reared monkeys (l/l = 26, l/s = 10) and 79 nursery-reared monkeys (l/l = 54, l/s = 25) were assessed on days 7, 14, 21, and 30 of life on a standardized primate neurobehavioral test designed to measure orienting, motor maturity, reflex functioning, and temperament. Both mother-reared and nursery-reared heterozygote animals demonstrated increased affective responding relative to l/l homozygotes. Nursery-reared, but not mother-reared, l/s infants exhibited lower orientation scores than their l/l counterparts. These results demonstrate the contributions of rearing environment and genetic background, and their interaction, in a nonhuman primate model of behavioral development.

The serotonin transporter (5-HTT) is most well known as the site of action of the serotonin reuptake inhibitors, which were initially developed as antidepressants, but now are the most widely used agents in the treatment of many additional neuropsychiatric and related disorders. The discovery that the gene that expresses the 5-HTT possesses a functional promoter-region polymorphism, which is associated with temperament and personality traits such as anxiety and negative emotionality as well as some behaviors, led to many studies examining this polymorphism in individuals with different neuropsychiatric disorders. The subsequent development of mice with a targeted disruption of the 5-HTT in our laboratory has provided an experimental model to examine the many consequences of diminished (in +/-, heterozygote mice) or absent (in -/-, homozygote knockout mice) function of the 5-HTT. The 5-HTT-deficient mice were also crossed with other knockout mice, allowing the study of multiple neurobiologic dysfunctions. As multiple genes are probably involved in the expression of complex behaviors such as anxiety, as well as neuropsychiatric disorders, these more genetically complex mice may more closely model disorders with complex etiologies. Thus, the combination of these comparative human and mouse studies may extend the opportunities to examine genetic alterations from a novel "bottom-up" approach [gene knockout or partial gene knockout in a combinational gene x gene x (yet unknown) gene approach], which is complementary to the traditional "top-down" genetic approach based upon studies of individuals with diagnosed neuropsychiatric disorders and their family members.

Idiopathic Parkinson's disease (PD) is a common neurodegenerative disorder with prominent motor symptoms. However, depression is common in PD, affecting about 40% of PD patients. Since there is extensive evidence of degeneration of serotonin (5HT) neurons and loss of the 5HT transporter (5HTT) in PD, we assessed whether a functional polymorphism in the promoter of the 5HTT gene (5HTT gene-linked polymorphic region, 5HTTLPR), which determines high or low 5HT uptake, is associated with depressive symptomatology in PD patients. We found that patients with the short allele of the 5HTTLPR had significantly higher scores on the Hamilton Depression Scale. A functional promoter polymorphism of the monoamine oxidase A (MAOA) gene showed no association. Thus, the 5HTTLPR but not the MAOA gene promoter-associated polymorphism may be a risk factor for depression in PD patients, while neither polymorphism increases the risk for development of Parkinson's disease itself.

Cocaine blocks uptake by neuronal plasma membrane transporters for dopamine (DAT), serotonin (SERT), and norepinephrine (NET). Cocaine reward/reinforcement has been linked to actions at DAT or to blockade of SERT. However, knockouts of neither DAT, SERT, or NET reduce cocaine reward/reinforcement, leaving substantial uncertainty about cocaine's molecular mechanisms for reward. Conceivably, the molecular bases of cocaine reward might display sufficient redundancy that either DAT or SERT might be able to mediate cocaine reward in the other's absence. To test this hypothesis, we examined double knockout mice with deletions of one or both copies of both the DAT and SERT genes. These mice display viability, weight gain, histologic features, neurochemical parameters, and baseline behavioral features that allow tests of cocaine influences. Mice with even a single wild-type DAT gene copy and no SERT copies retain cocaine reward/reinforcement, as measured by conditioned place-preference testing. However, mice with no DAT and either no or one SERT gene copy display no preference for places where they have previously received cocaine. The serotonin dependence of cocaine reward in DAT knockout mice is thus confirmed by the elimination of cocaine place preference in DAT/SERT double knockout mice. These results provide insights into the brain molecular targets necessary for cocaine reward in knockout mice that develop in their absence and suggest novel strategies for anticocaine medication development.

We report here that a truncated 5-HTT protein is produced in the neurons of the raphe, in serotonin transporter (5-HTT) knockout (KO) mice. The 5-HTT gene has exon 2 deleted and we found that one main transcript, shortened by 450 bp, is produced in these KO mice. The mutated 5-HTT protein is only recognized by antibodies against the C-terminal portion of 5-HTT. This protein is not functional as there is no high-affinity serotonin uptake in 5-HTT KO mice, in adults or during development. Conversely, low-affinity serotonin uptake was detected in vitro, and in dopaminergic neurons of the substantia nigra in vivo. The truncated 5-HTT, recognized by antibodies to the C-terminus, is present exclusively in the somatodendritic compartment of the raphe neurons instead of being exported to axons. As shown with confocal and electron microscopy, the truncated 5-HTT does not reach the plasma membrane and is essentially retained in the endoplasmic reticulum. However, this does not seem to trigger refolding or degradation responses, as no upregulation of the chaperone BiP or of the degradation signal ubiquitin was detected. Last, as observed in heterozygous mice, the presence of the truncated 5-HTT protein, although produced in large quantities, does not disturb the normal trafficking of the wild-type protein. This study therefore validates the 5-HTT KO model despite the occurrence of an incomplete translation, and brings novel information on the in vivo 5-HT uptake and cellular processing of an abnormal 5-HTT protein.

The consequences of the absence of 5-HT reuptake on the functional properties of 5-HT(1A) receptors were examined in the dorsal raphe nucleus and the hippocampus of knock-out mice lacking the serotonin transporter (5-HTT). Extracellular recordings showed that application of selective 5-HT reuptake inhibitors such as paroxetine and citalopram onto brainstem slices resulted in a concentration-dependent inhibition of 5-HT neuron firing in the dorsal raphe nucleus of wild-type 5-HTT+/+ mice, but not 5-HTT-/- mutants. By contrast, the 5-HT(1A) receptor agonists ipsapirone and 5-carboxamidotryptamine inhibited the discharge in both groups. However, the potency of these agonists was markedly decreased (by approximately 55- and approximately 6-fold, respectively) in 5-HTT-/- compared with 5-HTT+/+ animals. Similarly, intracellular recordings showed that the potency of 5-carboxamidotryptamine to hyperpolarize 5-HT neurons in the dorsal raphe nucleus was significantly lower in 5-HTT-/- than in 5-HTT+/+ animals. These data contrasted with those obtained with hippocampal slices in which 5-carboxamidotryptamine was equipotent to hyperpolarize CA1 pyramidal neurons in both mutant and wild-type mice. As expected from their mediation through 5-HT(1A) receptors, the effects of ipsapirone and 5-carboxamidotryptamine were competitively inhibited by the selective 5-HT(1A) antagonist WAY 100635 in both groups. These data showed that 5-HTT gene knock-out induced a marked desensitization of 5-HT(1A) autoreceptors in the dorsal raphe nucleus without altering postsynaptic 5-HT(1A) receptor functioning in the hippocampus. Similarities between these changes and those evoked by chronic treatment with 5-HT reuptake inhibitors emphasize the existence of regional differences in 5-HT(1A) receptor regulatory mechanisms.

The cross talk between dopaminergic and serotonergic systems in the brain has multiple neurophysiological and behavioral implications. Primary neuronal cultures of embryonic wild type (+/+) and serotonin transporter knockout (-/-) mice were used as a model to elucidate the possibility of plasticity at the level of serotonin uptake. Serotonergic neurons were identified in midbrain-hindbrain cultures of both wild type and knockout mice, using polyclonal anti-serotonin antibodies. Adding serotonin (10 microM) to wild type midbrain-hindbrain cultures increased the intensity of serotonin immunostaining, but did not change the number of serotonergic neurons. This increased intensity of serotonin staining was blocked by the serotonin transporter inhibitors fluoxetine and imipramine, but not with the dopamine transporter inhibitor nomifensine. In serotonin transporter knockout cultures, however, serotonin increased both the intensity of serotonin immunostaining and the number of serotonin positive neurons, and nomifensine decreased the number of serotonin-labeled neurons. Uptake of [3H]serotonin to wild type midbrain-hindbrain cultures was completely blocked by 1 microM fluoxetine, whereas nomifensine had a very small effect. In contrast, [3H]serotonin uptake to serotonin transporter knockout cultures, although very weak, was better inhibited by nomifensine than fluoxetine. The results imply that midbrain-hindbrain neuronal cultures of knockout mice, that do not express serotonin transporters, acquire the capacity to take up serotonin, apparently via dopamine transporters.

Serotonin (5-HT) is an important mediator of interactions between the nervous and immune systems. 5-HT signaling is regulated by the 5-HT transporter (5-HTT), which determines the magnitude and duration of serotonergic responses. Due to this important role, regulation of the 5-HTT by cytokines has been the focus of recent interest. A number of proinflammatory cytokines, including interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma, have been shown to upregulate the 5-HTT. In the present study we investigated the influence of interleukin-4 (IL-4), which acts as an anti-inflammatory cytokine in the central nervous system, on the 5-HTT. As a model system we used immortalized B lymphocytes, which not only express the 5-HTT, but also allow testing the co-modulatory influence of a recently described polymorphism in the 5-HTT gene promoter (5-HTTLPR) that is associated with anxiety- and depression-related behavioral traits. The results show that IL-4 induces a dose-dependent reduction of 5-HT uptake. This effect is preferentially seen in cell lines homozygous for the long, high-activity allele of the 5-HTTLPR. In conclusion, a picture of differential modulation of the 5-HTT by proinflammatory and anti-inflammatory cytokines is emerging, which may represent a fine-tuned mechanism to communicate the state of an immune response to the central nervous system.

The serotonin (5HT) transporter (5HTT) removes 5HT from the synaptic cleft and is thus critical to the control of serotonergic neurotransmission. Mice with a targeted inactivation of the 5HTT represent a novel and unique tool to study serotonergic system functioning. Because the release of 5HT is regulated by adenosine, we investigated 5HTT-deficient mice for possible adaptive changes of adenosine A(1) and A(2A) receptors. A(1) and A(2A) receptors were studied by means of quantitative autoradiography using the radioligands [3H]8-cyclopentyl-1,3-dipropylxanthine and [3H]CGS 21680, respectively. A comparison of 5HTT knockout versus control mice revealed upregulation of A(1) receptors in the dorsal raphe nucleus (DRN, +21%), but not in any of the serotonergic projection areas, and downregulation of A(2A) receptors in basal ganglia. The adaptive changes of A(1) and A(2A) receptors in 5HTT-deficient mice are likely to represent a compensatory neuroprotective effect mediated by the adenosinergic modulatory system. For comparison, these receptors were also studied in monoamine oxidase A (MAOA) knockout mice and in 5HTT/MAOA double knockout mice. 5HTT/MAOA double knockout mice showed adaptive changes of adenosine A(1) and A(2A) receptors similar to 5HTT knockout mice, while investigation of MAOA-deficient mice revealed an upregulation of A(2A) receptors, which may relate to a role of both MAOA and adenosine A(2A) receptors in anxiety.

By taking up serotonin (5-hydroxytryptamine, 5-HT) released in the extracellular space, the 5-HT transporter (5-HTT) regulates central 5-HT neurotransmission. Possible adaptive changes in 5-HT neurotransmission in knock-out mice that do not express the 5-HT transporter were investigated with special focus on 5-HT1A and 5-HT1B receptors. Specific labelling with radioligands and antibodies, and competitive RT-PCR, showed that 5-HT1A receptor protein and mRNA levels were significantly decreased in the dorsal raphe nucleus (DRN), increased in the hippocampus and unchanged in other forebrain areas of 5-HTT-/- vs. 5-HTT+/+ mice. Such regional differences also concerned 5-HT1B receptors because a decrease in their density was found in the substantia nigra (-30%) but not the globus pallidus of mutant mice. Intermediate changes were noted in 5-HTT+/- mice compared with 5-HTT+/+ and 5-HTT-/- animals. Quantification of [35S]GTP-gamma-S binding evoked by potent 5-HT1 receptor agonists confirmed such changes as a decrease in this parameter was noted in the DRN (-66%) and the substantia nigra (-30%) but not other brain areas in 5-HTT-/- vs. 5-HTT+/+ mice. As expected from actions mediated by functional 5-HT1A and 5-HT1B autoreceptors, a decrease in brain 5-HT turnover rate after i.p. administration of ipsapirone (a 5-HT1A agonist), and an increased 5-HT outflow in the substantia nigra upon local application of GR 127935 (a 5-HT1B/1D antagonist) were observed in 5-HTT+/+ mice. Such effects were not detected in 5-HTT-/- mice, further confirming the occurrence of marked alterations of 5-HT1A and 5-HT1B autoreceptors in these animals.

Recurrent panic attacks, anticipatory anxiety and phobic avoidance characterise panic disorder. The influence of genetic factors on liability to the disease has been the object of several linkage and association studies and appears to relate to an oligo- or polygenic rather than a monogenic mode of inheritance. Recently, an excess of high activity monoamine oxidase A (MAO-A) gene promoter alleles was found in female patients with panic disorder. An analysis of possible synergistic effects of the MAO-A gene promoter variant and the short serotonin transporter (5-HTT) gene promoter variant in panic disorder was performed in a German and an Italian sample (combined panic disorder n = 144, combined controls n = 175). There was no significant difference in odds ratios, suggesting that the observed increase of genetic liability by the long MAO-A gene promoter allele is not modified by the 5-HTT gene promoter polymorphism.

Hypoxia is a well-recognized stimulus for pulmonary blood vessel remodeling and pulmonary hypertension development. One mechanism that may account for these effects is the direct action of hypoxia on the expression of specific genes involved in vascular smooth muscle cell (SMC) proliferation. Previous studies demonstrated that the serotonin (5-hydroxytryptamine; 5-HT) transporter (5-HTT) mediates the mitogenic activity of 5-HT in pulmonary vascular SMCs and is overexpressed during hypoxia. Thus, 5-HT-related mitogenic activity is increased during hypoxia. Here, we report that mice deficient for 5-HTT (5-HTT(-/-)) developed less hypoxic pulmonary hypertension and vascular remodeling than paired 5-HTT(+/+) controls. When maintained under normoxia, 5-HTT(-/-)-mutant mice had normal hemodynamic parameters, low blood 5-HT levels, deficient platelet 5-HT uptake, and unchanged blood levels of 5-hydroxyindoleacetic acid, a metabolite of 5-HT. After exposure to 10% O(2) for 2 or 5 weeks, the number and medial wall thickness of muscular pulmonary vessels were reduced in hypoxic 5-HTT(-/-) mice as compared with wild-type paired controls. Concomitantly, right ventricular systolic pressure was lower and right ventricle hypertrophy less marked in the mutant mice. This occurred despite potentiation of acute hypoxic pulmonary vasoconstriction in the 5-HTT(-/-) mice. These data further support a key role of 5-HTT in hypoxia-induced pulmonary vascular SMC proliferation and pulmonary hypertension.

The serotonin transporter (5-HTT) regulates serotonergic neurotransmission and is thought to influence emotion. A 5-HTT-linked polymorphic region (5-HTTLPR) has two common variants, short (s) and long (l). We previously found population and within-family associations between the lower-expressing s allele and neuroticism, a trait related to anxiety, hostility, and depression, on a standard measure (the NEO Personality Inventory, Revised [NEO-PI-R]) in a primarily male population (n=505), and that the s allele was dominant. We investigated this association in a new sample (n=397, 84% female, primarily sib-pairs). The results robustly replicated the 5-HTTLPR neuroticism association, and the dominance of the s allele. Combined data from the two studies (n=902) showed a highly significant association between the s allele and higher NEO Neuroticism both across individuals and within families. Association between genotype and a related measure, Anxiety on the 16PF inventory, was replicated in the new population and within families in the combined sample. Association to another trait, estimated TPQ Harm Avoidance, was not replicated in the new sample but found only within the combined sibship group. Another association found in our original study, between the s allele and lower scores on NEO-PI-R Agreeableness, was also replicated and was more robust in the current and the combined samples. Associations between the functional 5-HTTLPR polymorphism were similar in women and men. These results help to define specific personality features reproducibly associated with 5-HTTLPR genotype. Such associations were strongest for traits defined by the NEO, enhancing the attractiveness of the five-factor personality model in genetic research on complex behavioral dimensions. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:202-216, 2000. Published 2000 Wiley-Liss, Inc.

The serotonin transporter (5-HTT) regulates serotonergic neurotransmission by determining the magnitude and duration of serotonergic responses. We have recently described a polymorphism in the 5-HTT gene promoter (5-HTTLPR) which influences the function of the 5-HTT and is associated with several psychiatric disorders. Immortalized B lymphocytes express the 5-HTT, and a B lymphocyte line has been shown to express the receptor for brain-derived neurotrophic factor, trkB. Since brain-derived neurotrophic factor (BDNF) is a specific growth and differentiation factor for serotonergic neurons, we assessed whether BDNF is able to modulate 5-HTT function in B lymphoblasts. Nerve growth factor (NGF), another neurotrophin which acts via the trkA receptor, was also studied. Eight immortalized B lymphoblast lines were generated and genotyped for the 5-HTTLPR. After treatment with BDNF or NGF, 5-HT uptake and proliferation of the cell lines were assessed. Two of the B cell lines showed a dose-dependent reduction of 5-HT uptake after exposure to BDNF. Both of these cell lines were homozygous for the long allele of the 5-HTTLPR. NGF did not influence 5-HT uptake or cellular proliferation in any of the cell lines. Thus, BDNF but not NGF may influence 5-HT uptake in some B lymphocytes. The fact that regulation of the 5-HTT was observed preferentially in cells of the long/long genotype indicates that presence of a short allele confers reduced regulatory capacity on the 5-HTT. In conclusion, B lymphoblasts represent a practical model for functional regulation of the 5-HTT by neurotrophins in serotonergic neurons.

The etiology of late-onset Alzheimer's disease (AD) and idiopathic Parkinson's disease (PD) is not known. In both disorders there is an extensive degeneration of serotonergic neurons, with corresponding losses of the serotonin (5HT) transporter (5HTT), which is responsible for the reuptake of 5HT from the synaptic cleft. An increasing body of evidence indicates that allelic variation of the 5HTT gene promoter (5HTT gene-linked polymorphic region, 5HTTLPR) determines high or low 5HT uptake in normal human brain. Association studies show that the low-activity allele of the 5HTTLPR is a risk factor for late-onset AD. In PD, the 5HTTLPR influences the risk of developing depression, a common symptom in PD patients. A compromised serotonergic system thus plays an important role in the pathophysiology of both AD and PD.

The aim of the present study was to determine whether alterations in 5-hydroxytryptamine (5-HT)(1A) receptors would be found in knockout mice lacking the serotonin transporter (5-HTT). Hypothermic and neuroendocrine responses to the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT) were used to examine the function of 5-HT(1A) receptors. Initial studies evaluated the dose-response and time course of 8-OH-DPAT-induced hypothermia and hormone secretion in normal CD-1 mice (the background strain of the 5-HTT knockout mice). 8-OH-DPAT dose-dependently produced hypothermic responses that peaked at 20 min postinjection. 8-OH-DPAT-induced hypothermia was blocked by the 5-HT(1A) antagonist WAY-100635. 8-OH-DPAT dose-dependently increased the concentrations of plasma oxytocin, corticotropin, and corticosterone. In the 5-HTT knockout (-/-) mice, the hypothermic response to 8-OH-DPAT (0.1 mg/kg s.c.) was completely abolished. Furthermore, 5-HTT-/- mice had significantly attenuated plasma oxytocin and corticosterone responses to 8-OH-DPAT. No significant changes in the hypothermic or hormonal responses to 8-OH-DPAT were observed in heterozygous (5-HTT+/-) mice. [(3)H]8-OH-DPAT- and [(125)I]MPPI [4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-iodobenzamido]ethyl] pip erazine]-binding sites in the hypothalamus and [(125)I]MPPI-binding sites in the dorsal raphe were significantly decreased in 5-HTT-/- mice. The results indicate that lack of the 5-HTT is associated with a functional desensitization of 5-HT(1A) receptor responses to 8-OH-DPAT, which may be a consequence, at least in part, of the decrease in density of 5-HT(1A) receptors in the hypothalamus and dorsal raphe of 5-HTT-/- mice.

The serotonin transporter (5-HTT) plays a key-role in the control of serotoninergic neurotransmission and is the target of some antidepressants. Possible adaptive changes in brain 5-HT2A receptors were investigated in knock-out mice that do not express the 5-HTT. Autoradiographic labeling of these receptors by the selective antagonist [3H]MDL 100,907 and saturation experiments with cortical membranes revealed: (1) a new localization of these receptors in the external field of striatum (possibly in striosomes); (2) regional variations in adaptive changes in the density of 5-HT2A receptors in 5-HT(-/-) mutants (-30-40% in the claustrum, cerebral cortex and lateral striatum; no significant change in the striatum core) as compared to wild-type mice.

Transcriptional activity of the human serotonin transporter gene (5HTT) is modulated by complex interaction of multiple genomic and cellular factors. Variability of a polymorphic repetitive element (5HTTLPR) is associated with anxiety, depression, and aggression-related traits and may influence the risk to develop affective spectrum disorders. 5HTTLPR variants display a unique DNA secondary structure that has the potential to regulate the transcriptional activity of the associated 5HTT promoter. The structure of the 5HTTLPR is also likely to precipitate a 381-bp somatic deletion in the 5HTT's promoter region [del(17)(q11.2)] that is observed in 20-60% of genomic DNA isolated from mononuclear blood cells and postmortem brain. The localization of the deletion breakpoints adjacent to identical putative signal sequences (CAGCC) suggests a V(D)J recombinase-like rearrangement event. In comparison with healthy controls, del(17)(q11.2)/wildtype sequence ratios showed a decrease of the deleted variant in recurrent unipolar depression. Our results also suggest that mosaicism of del(17)(q11.2) is likely to be regulated by tissue-specific as well as 5HTTLPR-dependent mechanisms. The findings confirm that the pericentric region of human chromosome 17 is highly unstable and furnishs additional evidence for intricate complexity of 5HTT regulation under physiological condition and in disease.

Serotonin (5-HT) is a prime candidate for studies of the interaction between the nervous and immune systems, since it is both an important neurotransmitter and released at high concentrations at sites of inflammation. Serotonergic neurotransmission is regulated by the 5-HT transporter (5-HTT), which determines the magnitude and duration of serotonergic responses. Since tumor necrosis factor alpha (TNF-alpha) and interleukin-6 are two inflammatory mediators that are central to the initiation of inflammation, we studied the impact of these cytokines on the 5-HTT. As model system we used a cell line which constitutively expresses the 5-HTT, namely the choriocarcinoma cell line JAR. We found that TNF-alpha enhances 5-HT uptake, with a doubling of the maximal velocity of uptake. Interleukin-6, on the other hand, had no effect. We thus show for the first time that the cytokine TNF-alpha modulates 5-HTT function. Furthermore, we propose a molecular mechanism for this effect. Since both 5-HT and TNF-alpha are elevated at sites of inflammation, TNF-alpha may act to renormalize 5-HT levels by way of its effect on the 5-HTT. This is especially important for the central nervous system, where the TNF-alpha effect shown here can aid in preventing disturbances of serotonergic neurotransmission.

Serotonin (5-HT) is an important regulator of morphogenetic activities during early central nervous system development, including cell proliferation, migration, and differentiation. The 5-HT transporter (5-HTT) plays a pivotal role in brain 5-HT homeostasis. It is also the initial target for both antidepressant drugs and drugs of abuse, some of which are potent neurotoxins. A polymorphism in the 5'-flanking regulatory region of the 5-HTT gene that results in allelic variation of 5-HTT expression is associated with anxiety-related personality traits and may influence the risk of developing affective disorders. Progress in 5-HTT gene inactivation studies are also changing views of the relevance of adaptive 5-HT uptake function in brain development and plasticity as well as processes underlying drug dependence and neurodegeneration. Despite evidence for a potential role of the 5-HTT in the integration of synaptic connections in the mammalian brain during development, adult life, and old age, detailed knowledge of the molecular mechanisms involved in these fine-tuning processes is just beginning to emerge. Integration of various strategies, including molecular genetic, transgenic, and gene transfer techniques, will allow elucidation of the 5-HTT's role in brain development, plasticity, and degeneration as well as in affective illness, drug abuse, and dementia.

Cocaine and methylphenidate block uptake by neuronal plasma membrane transporters for dopamine, serotonin, and norepinephrine. Cocaine also blocks voltage-gated sodium channels, a property not shared by methylphenidate. Several lines of evidence have suggested that cocaine blockade of the dopamine transporter (DAT), perhaps with additional contributions from serotonin transporter (5-HTT) recognition, was key to its rewarding actions. We now report that knockout mice without DAT and mice without 5-HTT establish cocaine-conditioned place preferences. Each strain displays cocaine-conditioned place preference in this major mouse model for assessing drug reward, while methylphenidate-conditioned place preference is also maintained in DAT knockout mice. These results have substantial implications for understanding cocaine actions and for strategies to produce anticocaine medications.

Background: We tested the hypothesis that a functional biallelic repetitive element in the 5' regulatory region of the human serotonin transporter gene (SLC6A4) confers susceptibility to serotonin-related personality traits underlying alcohol dependence with dissocial behavior., Methods: The association study was focused on 64 alcohol-dependent subjects with a dissocial personality disorder (according to ICD-10) who were derived from 315 German alcohol-dependent subjects. The Tridimensional Personality Questionnaire (TPQ) was applied to assess personality dimensions in 101 alcohol-dependent men, including 39 dissocial alcoholics., Results: Our association analyses revealed a trend towards a higher frequency of the short (S) allele of the SLC6A4 polymorphism in dissocial alcoholics compared to 216 German controls (chi 2 = 2.81, df = 1, p = 0.094). Dissocial alcoholics carrying the S/S genotype exhibited significant lower scores of harm avoidance compared to those lacking it (U-test, p = 0.015). Significantly higher novelty seeking scores were obtained in dissocial alcoholics carrying the S allele relative to those lacking it (U-test, p = 0.021)., Conclusions: Our tentative association findings in dissocial alcoholics suggest that the S allele of the 5' regulatory SLC6A4 polymorphism confers susceptibility to a temperamental profile of high novelty seeking and low harm avoidance that has been postulated to underlie dissocial (type-2) alcoholism according to Cloninger's neurogenetic theory of personality.

Central serotonergic neurotransmission has been implicated in the aetiology of ethanol tolerance and dependence. Cellular expression of the serotonin transporter and serotonin reuptake is modulated via a polymorphic, repetitive element in the 5'-flanking regulatory region of the serotonin transporter gene (5-HTTLPR). We report the association of the low-activity, short variant of the 5-HTTLPR with high ethanol tolerance among young adults in a case-control association study (n = 713). The low-activity 5-HTTLPR showed a significantly increased allele frequency (chi2 = 7.30; df = 2; P = 0.007) and genotype frequency among young adults (< or =26 years) with high ethanol tolerance homozygous for the short allele (chi2 = 7.58; df = 1; P = 0.02). The estimated odds ratio for the homozygous short variant compared to the homozygous long variant was 2.82 (95% CI 1.30-6.11). This indicates that the low-activity 5-HTTLPR may be involved in the neuronal mechanisms responsible for ethanol tolerance and dependence.

The sodium-dependent, high affinity serotonin [5-hydroxytryptamine (5-HT)] transporter (5-HTT) provides the primary mechanism for inactivation of 5-HT after its release into the synaptic cleft. To further evaluate the function of the 5-HTT, the murine gene was disrupted by homologous recombination. Despite evidence that excess extracellular 5-HT during embryonic development, including that produced by drugs that inhibit the 5-HTT, may lead to severe craniofacial and cardiac malformations, no obvious developmental phenotype was observed in the 5-HTT-/- mice. High affinity [3H]5-HT uptake was completely absent in 5-HTT-/- mice, confirming a physiologically effective knockout of the 5-HTT gene. 5-HTT binding sites labeled with [125I] 3 beta-(4'-iodophenyl)tropan-2 beta-carboxylic acid methyl ester were reduced in a gene dose-dependent manner, with no demonstrable binding in 5-HTT-/- mutants. In adult 5-HTT-/- mice, marked reductions (60-80%) in 5-HT concentrations were measured in several brain regions. While (+)-amphetamine-induced hyperactivity did not differ across genotypes, the locomotor enhancing effects of (+)-3, 4-methylenedioxymethamphetamine, a substituted amphetamine that releases 5-HT via a transporter-dependent mechanism, was completely absent in 5-HTT-/- mutants. Together, these data suggest that the presence of a functional 5-HTT is essential for brain 5-HT homeostasis and for 3,4-methylenedioxymethamphetamine-induced hyperactivity.

We have isolated and characterized the 5'-flanking regulatory region of the murine serotonin 5-HT transporter (5-HTT) gene. A TATA-like motif and several potential binding sites for transcription factors, including two AP1, several AP2 and AP4 binding sites, CCAAT and GC boxes (SP1 binding sites), a nuclear factor-kappaB, and a cyclic AMP response element-like motif, are present in the 5'-flanking region. A approximately 2.2-kb fragment (-2,143 to +51 with respect to the transcription start site), which had been fused to the luciferase reporter gene and transiently expressed in a 5-HTT-expressing cell line and in serotonergic raphe neurons derived from embryonic rat brainstem, displayed both constitutive and inducible promoter activity. Functional promoter mapping revealed two clusters of activating elements from bp -82 to -527 and bp -1,001 to -1,937. A cell/neuron-selective silencer element(s) is contained between bp -294 and -527. Our findings suggest that (1) the murine 5-HTT gene promoter is active in serotonergic raphe neurons but significantly repressed in neuronal cells from frontal cortex that do not express 5-HTT, (2) the information contained within approximately 0.5 kb of the 5'-flanking sequence is sufficient to confer its cell-selective expression, (3) the promoter responds to cyclic AMP- and protein kinase C-dependent induction, and (4) the expression of the 5-HTT is regulated by a combination of positive and negative cis-acting elements operating through a basal promoter unit defined by a TATA-like motif. Fusion of the 5-HTT gene promoter unit to a gene of choice may aid its cell-selective expression in transgenic strategies.

A possible dysregulation of serotonergic neurotransmission has been implicated in the aetiology of schizophrenic psychoses. In the present study we analysed allelic and genotypic variations of a recently described functional polymorphic region in the promoter of the human serotonin transporter gene (5-HTTLPR) and a variable tandem repeat (VNTR) in intron 2 of the 5-HTT gene. We investigated 413 unrelated individuals, 180 schizophrenic patients and 233 blood donors as controls. With regard to the 5-HTTLPR, both the schizophrenic and the control group did not significantly differ between genotype frequencies (chi2, p = 0.920) and allele frequencies (chi2, p = 0.836). The odds ratio for subjects with schizophrenia who were homozygous for the short allele was 1.04 (95% CI 0.59-1.84). No evidence of allelic association to specific schizophrenia subtypes was found. The 5-HTT associated VNTR also showed no significant differences between either the allelic or the genotypic distributions. Haplotype analysis revealed a significant overall linkage disequilibrium at a level of p = 0.00004. Our findings indicate that both polymorphisms are unlikely to play a substantial role in the genetic predisposition to schizophrenic disorders.

The norepinephrine transporter (NET) plays a critical role in brain norepinephrine homeostasis and is a target for antidepressants and drugs of abuse. We have analyzed the 5'-flanking regulatory region of the human NET gene (SLC6A2). Primer extension and 5' RACE revealed a single transcription start site, alternative splicing of exon 1 due to alternate splice donor usage, and a variable splice acceptor of intron 1. A TA-rich motif 35 bp upstream of the transcription start and several potential binding sites for transcription factors including a cAMP response element (CRE)-like motif are present in the 5'-flanking region. A 4.0-kb fragment, which had been fused to the luciferase reporter gene and transiently expressed in a NET+ cell line, displayed both constitutive and inducible promoter activity. Functional analysis by serial deletions revealed several clusters of cell-selective enhancer elements. Our findings indicate that (1) the NET gene promoter is active in NET-expressing cells and the information contained within approximately 4 kb of the 5'-flanking sequence is required to confer its cell-selective expression, (2) the expression of NET is regulated by a combination of positive cis-acting elements operating through a basal promoter defined by a TA-rich motif, and (3) the promoter responds to cAMP-dependent induction. Fusion of the human NET gene promoter to selected genes will facilitate their cell-selective expression in gene transfer strategies.

The high-affinity serotonin (5-HT) transporter (5-HTT) plays an important role in the removal of extracellular serotonin, thereby modulating and terminating the action of this neurotransmitter at various pre- and post-synaptic serotonergic receptors and heteroreceptors. In order to characterize the anatomical distribution of the 5-HTT in mouse brain, in situ hybridization histochemistry using 35S-labeled riboprobes was performed. These results were compared with 5-HTT binding site distribution as evaluated by [125I]RTI-55 autoradiography. High levels of 5-HTT mRNA were detected in all brain stem raphe nuclei, with variations in labeling among the various subnuclei. Those brain areas known to possess serotonergic cell bodies stained intensely for both 5-HTT mRNA and 5-HTT binding sites. In contrast to previous findings in rat brain, the highest densities of 5-HTT sites were found in areas outside the raphe complex, particularly in the substantia nigra, globus pallidus, and superior colliculi.

An association study was performed to elucidate the role of the serotonin transporter (5-HTT) gene as a susceptibility factor for autism as treatment of patients with antidepressant drugs which selectively target 5-HTT reduced autistic or concomitant symptoms, such as repetitive behavior and aggression, and ameliorate language use. Using the transmission/disequilibrium test (TDT) an analysis was done for a common polymorphism in the upstream regulatory region (5-HTTLPR), a VNTR in intron 2 of the gene and a haplotype of both loci in 52 trios fulfilling stringent criteria for autism and an extended group of 65 trios including patients showing no language delay in their first 3 years of life. A higher frequency and preferential transmission of the long allele of the 5-HTTLPR was observed, but the TDT gave a statistically significant value ( P = 0. 032) only for the extended patient group. This result is in contrast to a recent study by a US group presenting preliminary evidence for preferential transmission of the short allele of 5-HTTLPR in 86 trios. Both studies failed to reveal significant linkage disequilibrium between the VNTR in intron 2 of the gene and autism. In our study haplotype analysis of the 5-HTTLPR and the VNTR in intron 2 supplied evidence for an association of 5-HTT and autism in the stringent ( P = 0.069) and extended patient group ( P = 0.049). Overall, we were not able to replicate the findings of the first study on 5-HTT and autism and instead observed a tendency for association of the opposite genetic variant of the gene with the disorder. The implications for genetic variants of the serotonin transporter in the etiology of autism and possible subgroups of patients, therefore, needs clarification in further studies with other and larger patient samples.

The present study tested the hypothesis that the short, low activity variant of a biallelic polymorphism in the 5' regulatory region of the human serotonin transporter (5-HTT) gene confers susceptibility to severe alcohol dependence marked by severe withdrawal symptoms. Applying a phenotype-genotype strategy, our population-based association analysis included 216 German controls and an extreme sample of 103 severely affected alcoholics who were selected from 315 German alcohol-dependent subjects by a history of alcohol withdrawal seizure or delirium. The frequency of the short allele (S) was significantly increased in the severely affected alcoholics, compared with that in the controls (X2 = 3.87, df = 1, nominal p = 0.049). The post-hoc exploration indicated that this allelic association resulted exclusively from a significant excess of the S/S genotype in the severely affected alcoholics (p = 0.035), suggesting a recessively acting effect. Consistently, we found a weak but significant correlation (p = 0.013) between the frequency of the S/S genotype and severity of withdrawal symptoms (WDS): no WDS [18.3%, odds ratio (OR) = 1.16], vegetative WDS only (21.8%, OR = 1.44), and severe WDS with either withdrawal seizure only or delirium only (25.0%, OR = 1.69), and both withdrawal seizure and delirium (30.8%, OR = 2.30). Further studies are required to test whether the tentative genotype-phenotype relationship occurred by chance or reflects a real genotypic association between a recessively modifying effect of the short variant of the functional 5-HTT promoter polymorphism and alcohol withdrawal vulnerability.

Obsessive compulsive disorder (OCD) is a common illness, characterized by anxiety-provoking thoughts and the need to perform rituals. OCD is most commonly treated with a class of pharmacological agents known as serotonin reuptake inhibitors (SRIs). SRIs block the reuptake of serotonin (5-HT) into the presynaptic neuron, a process mediated by the serotonin transporter (5-HTT). The successful use of SRIs in OCD has led to the hypothesis that 5-HTT may play a pivotal role in the pathogenesis of OCD. We decided to study this hypothesis from a genetic perspective, because family and twin studies suggest that there is a strong genetic component to OCD. In addition, the sequence of the gene for 5-HTT is available, and a 44-bp insertion/deletion polymorphism has been detected in the promoter region of the gene. There is evidence that this polymorphism alters expression of the transporter protein. We typed 72 OCD patients and 72 matched controls, and found no statistically significant difference between the two groups (chi 2 = 4.319, P = 0.115, 2 d.f.). We observed however a trend towards increased homozygosity in the patient group. We also rated (retrospectively) the patients' clinical responses to SRIs. No association was observed between these ratings and the promoter region polymorphism in the serotonin transporter gene. Given the pharmacological evidence favoring a role for 5-HTT in OCD and SRI response, further genetic evaluation of the serotonin transporter in OCD is indicated.

To probe the hypothesis of a role for a functionally relevant 44 bp insertion/deletion of the serotonin transporter promoter in the aetiopathogenesis of panic disorder, we determined the allele frequency of the variant in two samples (combined n = 158) of panic disorder patients (DSMIII-R) and compared it with its allele frequency in two ethnically matched control samples (combined n = 169). The fact that no difference could be observed (x 2 analysis) argues against a major role for this serotonin transporter promoter polymorphism in the aetiopathogenesis of panic disorder.

By modulating the magnitude and duration of postsynaptic responses, carrier-facilitated serotonin (5-HT) transport into and release from the presynaptic neuron is central to the fine tuning of serotonergic neurotransmission. The 5-HT transporter (5-HTT) is the prime target for widely used antidepressants, psychostimulants, drugs of abuse and neurotoxins. We have isolated the gene encoding the murine 5-HTT and determined the sequence of all exons including adjacent intronic regions and approximately 3.6 kb of the 5'-flanking regulatory region. The murine 5-HTT gene is composed of 14 exons spanning approximately 34 kb. The single gene transcript after splicing is 2744 bp in length and it contains 186 bp of 5' untranslated region (5'-UTR) and 668 bp of 3'-UTR. A TATA-like motif and several potential binding sites for transcription factors including AP1, AP2, AP4, SP1 as well as CRE- and GRE-like motifs are present in the GC-rich 5'-flanking region. The characterization of murine 5-HTT cDNA and genomic organization will facilitate studies of 5-HT uptake function with molecular pharmacologic and transgenic strategies as well as investigations of its role in quantitative traits and psychiatric disorders.

We examined a deletion/insertion promoter polymorphism of the serotonin transporter gene, which confers an approximately 40% reduction in expression of the protein, in 196 subjects with late onset Alzheimer's disease (AD) and 271 controls. The frequency of the 484 bp low activity allele was elevated in the subjects with AD (p = 0.004), and an excess of the low activity genotype (30%) was also found in comparison with the controls (20%) (chi 2 = 7.16; p = 0.03). This association was unrelated to the age of the subjects or controls, or to epsilon 4 alleles of the ApoE gene. The odds ratio for the effect of the homozygous low activity genotype was 1.7 (95% CI 1.08-2.67), with a population attributable risk of 33% (95% CI 5-54%). These findings indicate that the low activity allele of the serotonin transporter is a risk factor for late onset AD.

Several lines of evidence implicate a role for the serotonergic system in body weight regulation and eating disorders. The magnitude and duration of postsynaptic responses to serotonin (5-HT) is directed by the transport into and release from the presynaptic neuron. Recently, a common polymorphism of a repetitive element in the region of the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) was identified that results in a system of two common alleles. The activity of the 5-HTT, as measured in in vitro assays and in human lymphoblastoid cell lines, is dependent on the respective genotype. We thus hypothesized that this polymorphism is relevant for weight regulation in general and is possibly involved in the etiology of anorexia nervosa (AN). Allele frequencies and genotypes were determined in a total of 385 unrelated obese children, adolescents and adults, 112 underweight subjects and 96 patients with AN. Furthermore, both parents of 98 obese children and adolescents and of 55 patients with AN, respectively, were genotyped, thus allowing to test for both association and linkage. The comparison of allele frequencies between obese and underweight probands provided no evidence for a major role of the 5-HTTLPR in weight regulation. Patients with AN had allele frequencies not significantly different to those observed for obese and underweight individuals.

Mood, emotion, and cognition are modulated by the serotonergic midbrain raphe system, which seems to be involved in the pathogenesis of psychiatric disorders like those of the affective spectrum. Since a dysregulation of serotonin transporter expression might be important in the course of those disorders, we isolated and cloned the 5'-regulatory region of the serotonin transporter gene, which is inducible by cAMP- and PKC-depended signal transduction pathways. Systematic screening for length variations and functional promoter analyses revealed a genetic polymorphism with allelic variation in transcriptional activity and protein expression. This 5-HTT gene polymorphism comprises a tandemly repeated sequence in the 5'-regulatory promoter region of the serotonin transporter gene. Recent population association studies demonstrated the 5-HTT gene promoter polymorphism accounting for 4-5% of population variation of anxiety-related behavioral traits.

Neurotransporters are high-affinity transport proteins located in the plasma membrane of both presynaptic nerve and glial cells that mediate the removal of neurotransmitters from the synaptic cleft or represent intracellular transport systems that concentrate neurotransmitters in synaptic vesicles. They comprise three subgroups, Na+/Cl(-)- or Na+/K(+)-dependent cell surface transporters and H(+)-dependent transporters associated with synaptic vesicles. The new insights into neurotransporter diversity provide the means for novel approaches of studying neurotransmitter uptake processes at the molecular level, such as substrate translocation and antagonist binding as well as regulation of gene expression, of intracellular trafficking, and of posttranslational modification. Moreover, modeling neurotransporter-related disorders and therapeutic strategies in genetically engineered animals are now feasible research strategies. Through an improved understanding of the modulation of neurotransporter function in the brain, it may be possible to identify the molecular factors underlying the etiopathogenesis and pathophysiology of neurodegenerative disorders. Due to their specificity for distinct neuronal systems, neurotransporters and their genes are potential targets for novel therapeutic strategies.

By conferring allele-specific transcriptional activity on the 5-HT transporter gene promoter in humans, the 5-HT transporter gene-linked polymorphic region (5-HTTLPR) influences a constellation of personality traits related to anxiety and increases the risk for neurodevelopmental, neurodegenerative, and psychiatric disorders. Here we have analyzed the presence and variability of the 5-HTTLPR in several species of primates including humans, and other mammals. PCR, Southern blot, and sequence analyses of the 5-HT transporter gene's 5'-flanking region in different mammalian species confirmed the presence of the 5-HTTLPR in platyrrhini and catarrhini (hominoids, cercopithecoids) but not in prosimian primates and other mammals. Since the 5-HTTLPR is unique to humans and simian primates, a progenitor 5-HTTLPR sequence may have been introduced into the genome some 40 Mio, years ago. In humans the majority of alleles are composed of either 14 or 16 repeat elements, while alleles with 18 or 20 repeat elements are rare. In contrast, great apes including orang-utan, gorilla, and chimpanzee display a high prevalence of alleles with 18 and 20 repeat elements. In hominoids all alleles originate from variation at a single locus (polymorphic locus 1). In the 5-HTTLPR of rhesus monkeys (rh5-HTTLPR) we found an alternative locus for length variation (polymorphic locus 2) generated by a 21 bp insertion/deletion event. The existence of a distinct biallelic variation of the 5-HTTLPR in rhesus monkeys but similar allele and genotype frequencies in this species and humans supports the notion that there may be a relationship between functional 5-HT transporter expression, anxiety-related traits, and the complexity of socialization in human and non-human primate populations.

The serotonin transporter (5-HTT) is a candidate locus for aetiological involvement in affective disorders. Biochemical studies in suicides and depressed patients suggest that 5-HT uptake function is frequently reduced in affective illness. Furthermore, 5-HTT is targeted by widely used antidepressant drugs such as fluoxetine. We have performed an association study of a short variant of the 5-HTT-linked polymorphic region (5-HTTLPR), which restricts transcriptional activity of the 5-HTT promoter leading to low functional expression of the 5-HTT, in 454 patients with bipolar or unipolar affective disorder and 570 controls, derived from three European Centres (London, Milan and Würzburg). In all three centres, the frequency of the low activity allele was higher in patients than in controls (50% vs 45% in London, 45% vs 43% in Milan, 47% vs 40% in Würzburg). Although these differences were not individually significant, a stratified analysis of all three samples gave a significant overall odds ratio of 1.23 (95% confidence interval 1.02-1.49, P = 0.03). The excess of the homozygous low-activity genotype among the patients was even greater (odds ratio 1.53, 95% confidence interval 1.04-2.23, P = 0.02), suggesting partial recessively of the low-activity allele. Given the functional role of 5-HTT, our findings suggest that 5-HTTLPR-dependent variation in functional 5-HTT expression is a potential genetic susceptibility factor for affective disorders. If this finding is replicated, further work on genetic variants with low 5-HTT activity may facilitate the differential diagnosis of affective disorders, the assessment of suicidal behaviour, and the prediction of good clinical response to antidepressants.

Transporter-facilitated uptake of serotonin (5-hydroxytryptamine or 5-HT) has been implicated in anxiety in humans and animal models and is the site of action of widely used uptake-inhibiting antidepressant and antianxiety drugs. Human 5-HT transporter (5-HTT) gene transcription is modulated by a common polymorphism in its upstream regulatory region. The short variant of the polymorphism reduces the transcriptional efficiency of the 5-HTT gene promoter, resulting in decreased 5-HTT expression and 5-HT uptake in lymphoblasts. Association studies in two independent samples totaling 505 individuals revealed that the 5-HTT polymorphism accounts for 3 to 4 percent of total variation and 7 to 9 percent of inherited variance in anxiety-related personality traits in individuals as well as sibships.

Dysfunctions in serotonergic pathways may underlie several psychiatric disorders. The reuptake of serotonin (5-HT) from synaptic terminals is mediated by a specific transporter (5-HTT). Genetic variation in the gene coding for the 5-HTT protein might be involved in the predisposition to psychiatric disorders. A systematic screening of the whole coding sequence of the 5-HTT gene in mood disorder (MD) and obsessive-compulsive disorder (OCD) patients, as well as in healthy controls, using PCR and denaturing gradient gel electrophoresis (DGGE) revealed the presence of two mutations. The first was in intron 4, and the second was a C-->A transversion leading to an amino-acid exchange (Leu-->Met) in position 255 of the deduced protein sequence. No further occurrence of this substitution was found in an extended sample of patients and controls. Therefore, structural modifications of the 5-HTT gene do not seem to play either a major or minor role in the genetic predisposition to MD or OCD.