Monoamine Transporter Pathologies 175 site just above the extracellular gate (Singh et al., 2007; Zhou et al., 2007). This structure may reveal a similar binding site for the mammalian monoamine trans- porters, although some have questioned if the TCA binding site of LeuT Aa is likely to be reflective of such a site in the monoamine transporters (Henry et al., 2007; Rudnick,2007). 1.5 Vesicular Monoamine Transporters Although plasma membrane monoamine transporters are responsible for the reup- take of neurotransmitters from the synapse, vesicular monoamine transporters (VMAT) sequester monoamines into synaptic vesicles in preparation for fusion with the plasma membrane and release into the synapse (Schuldiner et al., 1995). Vesicular uptake is coupled to a proton gradient across the vesicle membrane rather than the sodium gradient used with the plasma membrane transporters (Schuldiner et al., 1995). These vesicular transporters are not neurotransmitter- specific; rather, they transport the monoamines nonselectively (Johnson, Jr., 1988; Henry et al., 1998). VMAT is predicted to have similar membrane topology to the plasma mem- brane monoamine transporters, although they do not share homologous sequences (Erickson et al., 1992). Hydrophobicity studies predict 12 TMHs with amino and carboxy termini located in the cytoplasm (Erickson et al., 1992). The large extra- cellular loop between TMHs III and IV of the plasma membrane transporters is located between TMH I and II in VMAT (Erickson et al., 1992). VMAT1 is located in the neuroendocrine cells of the adrenal medulla and intestinal tract, whereas VMAT2 is found in monoaminergic neurons of the central nervous system (Erickson et al., 1996). Because VMAT regulates the level of cytosolic monoamines, researchers have examined a role for VMAT in disease states. Although no direct pathological links to aberrant VMAT function have been described, altered dopamine regulation can lead to drug addiction, Parkinson’s disease, and schizophrenia (Mazei-Robison et al., 2008). Psychostimulants have been demonstrated to affect dopaminergic signaling by altering DAT and VMAT function (Fleckenstein et al., 2009). Such alterations can be neurotoxic and may provide a role f or the monoamine transporters in Parkinson’s disease (Fleckenstein et al., 2009). 2 Regulation of Plasma Membrane Monoamine Transporters Plasma membrane monoamine transporters serve an important regulatory role in maintaining appropriate levels of monoamines in the synapse (Torres et al., 2003a). Aberrant regulation of transporter expression and function has been implicated in several disease states (Howell and Kimmel, 2008). The monoamine transporters are regulated by interaction with a number of substrates and antagonists with vary- ing affinities for the transporters at the plasma membrane (Sulzer et al., 1995; Gutman and Owens, 2006; Fleckenstein et al., 2007). In addition to transporting 176 N.R. Sealover and E.L. Barker their respective neurotransmitters, the monoamine transporters can lose substrate selectivity under certain conditions. DAT and NET can each transport dopamine and norepinephrine (Giros et al., 1994), and SERT displays an increased preference for dopamine at elevated temperatures (Saldana and Barker, 2004). Amphetamines such as methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”) are also substrates of the monoamine transporters, as are some neurotox- ins such as 1-methyl-4-phenylpyridinium (MPP + ) (Torres et al., 2003a). In addition, the monoamine transporters are influenced by several classes of antagonists, including cocaine and antidepressants (Torres et al., 2003a). Monoamine transporter regulation can occur by altering transporter surface expression. Monoamine transporters contain sites for potential phosphorylation in the cytoplasmic loops and the carboxy terminal region (Jayanthi and Ramamoorthy, 2005). Samuvel and colleagues demonstrated that p38 mitogen-activated protein kinase (MAPK) regulates SERT by inhibiting cell surface expression (Samuvel et al., 2005). Treatment of cells and synaptosomes with the PKC activator, phor- bol 12-myristate13-acetate (β-PMA) reduces monoamine transport capacity (V max ) without altering substrate affinity (K m ) (Samuvel et al., 2005). Other agents that maintain the phosphorylated state of the monoamine transporters such as phos- phatase inhibitors also reduce V max (Vaughan et al., 1997; Ramamoorthy et al., 1998; Jayanthi et al., 2004; Apparsundaram et al., 1998b, a). The phosphatase inhibitor, okadaic acid, downregulates DAT, NET, and SERT activity (Ramamoorthy et al., 1998). These studies suggest that phosphorylation of monoamine t ransporters impairs plasma membrane expression. SERT and protein phosphatase 2A (PP2A) form a complex that is regulated by p38 MAPK activation (Zhu et al., 2005). This complex is inhibited by PP2A inhibitors and PKC activators (Bauman et al., 2000). This complex is stabilized in the presence of the substrate 5-HT (Bauman et al., 2000). These studies provide a mechanism for the regulation of transporter function through the interaction of SERT with PP2A. Monoamine transporter function is also regulated by glycosylation. The large extracellular loop between TMH III and TMH IV of the monoamine transporters contains consensus sites for glycosylation (Melikian et al., 1994, 1996). The glyco- sylated form of the transporter is the mature form that undergoes insertion into the plasma membrane (Sitte et al., 2004). Functional monoamine transporters are predicted to form oligomers (Milner et al., 1994; Jess et al., 1996; Kilic and Rudnick, 2000). One study reports the exis- tence of a dimer of dimers (Kilic and Rudnick, 2000). This tetramer is proposed to be the functional form that exists in the plasma membrane (Kilic and Rudnick, 2000). A leucine heptad repeat in TMH II and a glycophorin-like motif in TMH VI are thought to play a role in stabilizing the oligomeric form of DAT (Torres et al., 2003b). The formation of SERT dimers results from a putative interaction involving TMH XI and TMH XII (Just et al., 2004). The monoamine transporters are also regulated by a feedback mechanism that involves monoamine autoreceptors located on the presynaptic cell membrane (Hjorth et al., 2000; Schmitz et al., 2002; Garcia et al., 2004). These autoreceptors detect the levels of various monoamines in the synapse and modulate the release of Monoamine Transporter Pathologies 177 monoamines to keep appropriate levels of neurotransmitter in the synapse (Hjorth et al., 2000; Schmitz et al., 2002; Garcia et al., 2004). The exact mechanism of this feedback loop is unknown (Hjorth et al., 2000; Schmitz et al., 2002; Garcia et al., 2004). The autoreceptors are the D2 short isoform (D2 s), α 2A , and 5-HT 1B recep- tors for dopamine, norepinephrine, and serotonin, respectively (Xie et al., 2008). The feedback loop may also be controlled by the trace amine-associated receptor 1 (TAAR1). TAAR1 is a G protein-coupled receptor that is activated by the bio- genic monoamines, trace amines, and psychostimulants (Borowsky et al., 2001). Xie and colleagues demonstrated the regulation of DAT by TAAR1 and the regula- tion of TAAR1 signaling by D2 s (Xie and Miller, 2007; Xie et al., 2007). Similar studies have been conducted with NET and SERT to show the regulation of these transporters by TAAR1 and monoamine autoreceptors (Xie et al., 2008). 3 Transporter Gene Polymorphisms Several genetic polymorphisms have been identified for the genes encoding the monoamine transporters. A brief review of these genetic variations and possible associations with disease states is presented below and in Table 1. A comprehensive review by Hahn and Blakely examines the impact of genetic variations of the SLC6 gene family (Hahn and Blakely, 2007). Table 1 Summary of identified polymorphisms for NET, DAT, and SERT Polymorphism Effect of Polymorphism Possible Pathological Associations NET A457P Impaired transport, decreased cell surface expression Orthostatic intolerance, increased heart rate F528C Elevated transport, decreased TCA potency High blood pressure –3801 (A/T) Transcription factor-based repression of NET expression ADHD DAT A559V Increased Na + sensitivity, spontaneous DA efflux ADHD, bipolar disorder 3 untranslated VNTR (480 bp) Unknown ADHD SERT I425V Increased transport, increased V max , decreased K m OCD, Asperger’s syndrome 5-HTTLPR (s) Reduced gene transcription OCD, ADHD, depression VNTR Regulates transcription No known links 3.1 NET A number of nonsynonymous single nucleotide polymorphisms (SNPs) that result in single amino acid substitutions have been identified for the monoamine transporters. 178 N.R. Sealover and E.L. Barker The hNET SNP A457P was discovered in a familial form of orthostatic intolerance (Hahn et al., 2003; Shannon et al., 2000). The A457P allele was found to be associ- ated with increased heart rate and plasma norepinephrine levels (Hahn et al., 2003). Molecular studies demonstrate t hat hNET A457P has severely impaired transport function and decreased cell surface expression, revealing a mechanism for impaired hNET function and cardiovascular disease (Hahn et al., 2003). Approximately 20 more coding region SNPs have been identified for hNET, primarily associated with altered psychiatric and cardiovascular phenotypes (Hahn et al., 2005). The precise functional role of many of these variants remains largely undefined. However, the hNET variant F528C was discovered in patients with high blood pressure (Hahn et al., 2005). Hahn and colleagues found the hNET variant to have elevated trans- port levels, decreased tricyclic antidepressant potency, and an insensitivity to PKC downregulation by β-PMA (Hahn et al., 2005). In addition to the potential significance of coding region SNPs, variations in the hNET promoter region have also been identified (Kim et al., 2006). The substitution of adenine to thymine at –3081 has been linked to ADHD (Kim et al., 2006). The thymine substitution establishes a palindromic E2-box motif that binds the neural- expressed repressors of transcription, Slug and Scratch (Kim et al., 2006). Slug and Scratch bind the E2-box motif and repress SLC6A2 promoter activity only when the thymine substitution is present. These data suggest that the –3081(A/T) polymor- phism, resulting in transcription factor-based repression of SLC6A2, may increase the risk of ADHD development (Kim et al., 2006). 3.2 DAT The presence of SNPs is not unique to NET. Studies have revealed variants of DAT and SERT as well. A rare DAT coding SNP, A559V, has been identified in two male children diagnosed with ADHD (Mazei-Robison et al., 2008) and a female with bipolar disorder (Grunhage et al., 2000). Cellular studies have demon- strated an increased sensitivity to intracellular sodium and increased DA efflux for hDAT A559V in the absence of efflux-inducing amphetamines (Mazei-Robison et al., 2008). Homology modeling based on LeuT Aa , places A559 at the extracel- lular end of TMH 12 (Mazei-Robison et al., 2008). Studies have implicated TMHs 11 and 12 as forming the interface for monoamine transporter dimerization (Just et al., 2004). Dimerization is known to be important for serotonin efflux (Seidel et al., 2005). The increased dopamine efflux observed for A559V may be due to impairment of transporter dimerization (Mazei-Robison et al., 2008). Interestingly, Chen and colleagues demonstrated that mutating S528 to alanine in DAT TMH 11 results in increased dopamine efflux (Chen and Justice, 2000). These find- ings suggest a mechanism by which altered DA efflux may be linked to disease states. The DAT gene is located on chromosome 5 and contains a variable number tan- dem repeat (VNTR) polymorphism in the 3 -untranslated region. This VNTR is composed of 40 bp repeats that commonly contain nine or ten copies. Multiple Monoamine Transporter Pathologies 179 investigations have found a link between ADHD and the 480 bp VNTR (Barr et al., 2001; Chen et al., 2003; Cook, Jr. et al., 1995; Curran et al., 2001; Daly et al., 1999; Gill et al., 1997; Waldman et al., 1998). Researchers are unclear if the num- ber of repeats in the 3 untranslated VNTR directly controls the expression level of the DAT gene or if the allele containing this VNTR is in linkage disequilib- rium with functional DNA variants that contribute to the ADHD phenotype (Barr et al., 2001). 3.3 SERT SERT gene variants have been implicated in neuropsychiatric disorders. Ozaki and colleagues identified the presence of an I425V coding region SNP in some individ- uals affected with obsessive compulsive disorder (OCD) and Asperger’s syndrome (Ozaki et al., 2003). Studies in cultured cells found the I425V mutation to cause an increased rate of transporter activity with an increase in V max and decrease in K m (Kilic et al., 2003). Cell surface expression was unchanged for the mutant. The ele- vated transport is thought to be caused by altered cGMP-dependent protein kinase activity (PKG). The I425V mutation results in constitutive activation of SERT sim- ilarly to the way nitric oxide stimulates wild-type SERT via a PKG-dependent pathway (Kilic et al., 2003). The stimulation of SERT by cGMP is disrupted in the I425V mutant, although the exact mechanism by which this occurs remains unknown. Thr 276 is predicted to be in the second intracellular loop, between TMH IV and V, and is the site of PKG phosphorylation on SERT (Ramamoorthy et al., 2007). Ile 425 is predicted to reside in the middle of TMH VIII near putative sub- strate and inhibitor binding sites. It is unclear if the I425V mutation activates the transporter in a manner that makes Thr 276 phosphorylation irrelevant, or if this mutation indirectly increases the level of Thr 276 phosphorylation by interfering with the activity of a phosphatase (Zhang et al., 2007). Two common polymorphisms have also been reported in the promoter region of the SERT gene. The first is the insertion or deletion of a 44 bp sequence that results in a long (L) or short (S) allele termed the 5-HTTLPR (Lesch et al., 1996). The S variant displays threefold reduced gene transcription, leading to decreased transporter expression and 5-HT uptake (Lesch et al., 1996). Patients with major depression who are homozygous for the long allele (L/L) or heterozygous (L/S) respond better to treatment with the SSRIs fluvoxamine and paroxetine than those homozygous for the short allele (S/S) (Lesch et al., 1996; Zanardi et al., 2000). The S allele has been associated with an increased risk of depression, obsessive- compulsive disorder, and ADHD (Torres et al., 2003a). The second type of promoter polymorphism is a VNTR in the second intron composed of 17-bp repeats (Ogilvie et al., 1996). Ten and twelve sets of repeats are most common (Lesch et al., 1994). Studies with embryonic stem cells and transgenic embryos implicate the VNTR as playing a role in the regulation of tran- scription, although no definitive links are known between this VNTR and disease states (Torres et al., 2003a). 180 N.R. Sealover and E.L. Barker 4 Addiction 4.1 Psychostimulant Addiction The rewarding and reinforcing effects of psychostimulants appear to rely primarily on the dopamine system, although studies have demonstrated the ability of sero- tonin and norepinephrine systems to produce behavioral and neurochemical effects in response to psychostimulants (Howell and Kimmel, 2008). DAT and VMAT2 are critical players in the regulation of dopamine levels in the synapse and cytosol, respectively. The GABAergic system can regulate dopaminergic signaling by con- trolling the firing rate of dopamine neurons (Churchill et al., 1992; Steffensen et al., 1998). Psychostimulants exert their effects by increasing levels of extracellular neurotransmitter. Psychostimulants are classified as uptake inhibitors or releasers. Cocaine is an example of an uptake inhibitor (Table 2). Cocaine exerts its effects by binding to DAT, NET, or SERT. This binding prevents the transport of neurotrans- mitter, resulting in increased synaptic neurotransmitter levels. Amphetamines such as MDMA are classified as releasers. They are substrates of the monoamine trans- porters. Releasers reverse the direction of transport from inward to outward, leading to an increase in the levels of neurotransmitter in the synaptic cleft (Fleckenstein et al., 2007; Rothman and Baumann, 2003). Repeated exposure to psychostimulants can modify neurotransmitter systems and result in tolerance or increased sensitivity. This exposure alters the effects of Table 2 Structures of psychostimulants and K i values in μmol/L for inhibition of [ 3 H] 5-HT, [ 3 H] NE, and [ 3 H] DA uptake at hSERT, hNET, and hDAT, respectively Structure Name hSET hNET hDAT Cocaine 0.03 0.48 ± 0.05 0.23 ± 0.03 MDMA 0.73 1.19 ± 0.13 8.29 ± 1.67 Amphetamine 3.84 0.07 ± 0.01 0.64 ± 0.14 H N O O Methylphenidate 10.71 0.10 ± 0.01 0.06 ± 0.01 0.74± 132.43 ± 38.46 ± 2.41 ± Data were obtained in Intestine 407 cells transfected with hSERT, hNET, or hDAT (Han and Gu, 2006). Monoamine Transporter Pathologies 181 drugs on brain neurochemistry and behavior, ultimately disrupting the neurobio- logical regulation of functions related to addiction (Howell and Kimmel, 2008). Interestingly, conflicting studies have reported that cocaine administration in rodents may result in increased, decreased, or unaltered DAT, D1, and D2 receptor levels (Pilotte et al., 1994; Wilson et al., 1994; Claye et al., 1995; Boulay et al., 1996; Tella et al., 1996; Letchworth et al., 1997; Letchworth et al., 1999). Repeated cocaine use has been shown to increase DAT activity in humans (Mash et al., 2002). The ini- tial increase in extracellular dopamine after cocaine administration is thought to result in increased DAT function as a compensatory mechanism. Increased DAT function in turn leads to reduced levels of extracellular dopamine even in the absence of cocaine. This cycle of altered synaptic dopamine levels and DAT func- tion is thought to contribute to the addictive properties of psychostimulants such as cocaine. Despite significant public health concerns surrounding psychostimulant abuse, currently no effective pharmacotherapies exist (Howell and Kimmel, 2008). To date, treatment for cocaine addiction has been the most widely studied of all psy- chostimulants. Researchers have examined potential benefits of antidepressants and dopamine receptor agonists and antagonists for cocaine addiction with lit- tle success. The TCA desipramine was reported to be an effective treatment in outpatient clinical trials (Levin and Lehman, 1991). Further clinical trials were not able to confirm this effectiveness (Arndt et al., 1992; Campbell et al., 1994). Similarly, treatment with the selective serotonin reuptake inhibitor (SSRI) flu- oxetine, appeared initially promising (Walsh et al., 1994), but further studies were unable to demonstrate effectiveness over placebo controls (Batki et al., 1996; Grabowski et al., 1995). Clinical studies with the D2-like receptor agonist bromocriptine have yielded inconclusive results (Gorelick, 1992). Studies target- ing GABAergic transmission have shown recent promise (Sofuoglu and Kosten, 2005). Treatment with baclofen, an antispasticity medication and GABA B receptor agonist, has resulted in increased cocaine abstinence in cocaine-addicted patients (Shoptaw et al., 2003). Similarly, the GABA transporter inhibitor tiagabine that is used for treating epilepsy has been shown to reduce cocaine dependence (Gonzalez et al., 2003). These studies implicate the GABAergic system as a promising tar- get for the development of useful pharmacotherapies for the treatment of cocaine addiction. 4.2 Alcoholism Alcoholism i s characterized by the development of tolerance, craving, and with- drawal (Heinz et al., 2004). Repeated exposure to alcohol results in neuroadaptive changes in the central dopaminergic and serotonergic systems (Heinz et al., 2004). Several studies have directly implicated DAT and SERT in alcoholism. A reduction in SERT expression was found in a sample of alcoholic patients (Heinz et al., 1998) and a high frequency of the short 5-HTTLPR was observed in alcoholic patients (Hammoumi et al., 1999). Studies in rodents demonstrated an ethanol-induced 182 N.R. Sealover and E.L. Barker release of dopamine that reinforced the mesolimbic reward system (Mereu et al., 1984; Di Chiara and Imperato, 1988). A recent study by Hillemacher and colleagues found significant hypermethylation of the DAT promoter in alcohol-dependent patients compared to healthy control subjects (Hillemacher et al., 2009). They pro- posed that ethanol consumption in alcoholics may lead to reduced craving due to hypermethylation-induced downregulation of genes including DAT (Hillemacher et al., 2009). Hypermethylation of the DAT promoter is thought to inhibit gene transcription, leading to reduced DAT expression and increased levels of synaptic dopamine. The mechanism for DAT promoter methylation in response to ethanol consumption is unknown, although the long-term regulation of gene expression by epigenetic mechanisms such as DNA methylation has been suggested as playing a role in the pathophysiology of several psychiatric disorders (Hillemacher et al., 2009). 5 Anxiety and Depression Alterations in the serotonergic and noradrenergic systems are well established in the pathophysiology of mood disorders, including anxiety and depression. Studies have demonstrated a linkage between the short 5-HTTLPR and psychiatric conditions (Olivier et al., 2008). Anxiety disorders include panic, phobias, obsessive compul- sive disorder, and generalized anxiety disorder (GAD) (Keller et al., 2006). These disorders are often treated by blocking NET with compounds such as reboxetine or atomoxetine (Morilak and Frazer, 2004). Chronic treatment with reboxetine or desipramine in rats has been shown to decrease NET binding sites (Gould et al., 2003; Frazer and Benmansour, 2002). Anxiety disorders carry periods of high emo- tional distress accompanied by physiological hyperarousal (Keller et al., 2006). Keller and colleagues demonstrated that NET-deficient mice respond to stress- inducing environments with heightened autonomic cardiovascular response (Keller et al., 2006). This cardiovascular response is consistent with a NET deficiency linked to increased blood pressure and heart rate due to anxiety and fear-inducing stimuli (Keller et al., 2006). Several theories have attempted to explain the pathology of depression. One of these theories is the monoamine theory of depression (Heninger et al., 1996). This theory proposes that impaired monoaminergic function is the central basis behind depression. Serotonin and norepinephrine are the two monoamines that have been primarily implicated in the disease. Pharmacological treatment of depres- sion has focused on increasing synaptic levels of these two neurotransmitters (Table 3). The first class of antidepressants was developed in the early 1950s with the dis- covery of an antitubercular drug iproniazid that possesses mood-elevating properties (Nutt, 2002). Iproniazid is a monoamine oxidase inhibitor (MAOI). Monoamine oxi- dase is the enzyme that breaks down serotonin, dopamine, and norepinephrine. The inhibition of monoamine oxidase increases levels of monoamines in the synapse. Monoamine Transporter Pathologies 183 Table 3 Structures of antidepressants and K i values in nmol/L for [ 3 H] 5-HT or [ 3 H] NE inhibition at hSERT and hNET, respectively Structure Name hSERT hNET Desipramine 163 ± 5 3.5 ± 0.6 N (CH 2 ) 3 N(CH 3 ) 2 Imipramine 20 ± 2 142 ± 8 Amitriptyline 36 ± 1 102 ± 9 Nortriptyline 279 ± 20 21 ± 0.77 Paroxetine 0.83 ± 0.06 328 ± 25 Citalopram 8.9 ± 0.7 30,285 ± 1600 Fluoxetine 20 ± 2 2186 ± 142 Sertraline 3.3 ± 0.4 1716 ± 151 Venlafaxine 102 ± 9 1644 ± 84 Data were obtained in HEK-293 cells transfected with hSERT or hNET (Owens et al., 1997). 184 N.R. Sealover and E.L. Barker Potentially life-threatening interactions with foods containing tyramine and tryp- tophan led to the disuse of these drugs and the development of different classes of antidepressants. Since the discovery of the first MAOI, TCAs, SSRIs, serotonin– norepinephrine reuptake inhibitors (SNRIs), and some atypical antidepressants such as buproprion have been used in the treatment of depression. Except for the atypical antidepressants, the aforementioned classes of antidepressants increase synaptic lev- els of s erotonin or norepinephrine by inhibiting SERT or NET, r espectively (White et al., 2005). White and colleagues provide a comprehensive review of the antide- pressants in each of these classes (White et al., 2005). Despite the development of new classes of antidepressants, the effectiveness of these therapeutics remains no better than the MAOIs and patient compliance remains low (Song et al., 1993). Inhibiting SERT and NET rapidly increases synaptic neurotransmitter levels, but the maximal clinical effect is not observed until after several weeks of treatment (Gelenberg and Chesen, 2000). As with chronic administration of NET inhibitors, long-term exposure to SERT inhibitors results in decreased SERT surface expres- sion (Benmansour et al., 1999, 2002). These decreased SERT and NET levels may help to explain the lapse in time from the initial administration of antidepressants to their maximum clinical efficacy. 6Autism Autism is a neurodevelopmental disorder that appears in early childhood and results in severely impaired behavioral functions (Folstein and Rosen-Sheidley, 2001). Children with autism display poor social interactions, impaired speech develop- ment, and an interest in repetitive activities (Folstein and Rosen-Sheidley, 2001). Autism is recognized as a heritable disorder (Macdonald et al., 1989), although twin-based studies indicate that the disorder is not always inherited (Murphy et al., 2000). Research indicates that autism is linked to neuronal disorganization and the disarrangement of neurotransmitter pathways (Pardo and Eberhart, 2007). The serotonin hypothesis of autism describes the importance of genes that regu- late the serotonin system. In particular, genes that control serotonin metabolism and neurotransmission have received much attention (Cook and Leventhal, 1996; Buitelaar and Willemsen-Swinkels, 2000). The serotonin hypothesis of autism is supported by an improvement in behavioral functions in autistic patients receiv- ing treatment with SSRIs (Hollander et al., 2003) or 5-HT2 receptor antagonists (Pardo and Eberhart, 2007). A recent study by Makkonen and colleagues demon- strates reduced SERT binding capacity in the medial frontal cortex of children with autism (Makkonen et al., 2008). This study used single-photon emission computed tomography (SPECT) to analyze the binding of [ 123 I] labeled N-(2-fluoroethyl)-2β- carbomethoxy-3β-(4-iodophenyl)-nortropane, ([ 123 I] nor-β-CIT) to SERT and DAT. A significant decrease in SERT binding, but not DAT binding was demonstrated. Whereas a number of factors likely contribute to the pathology of autism, a signif- icant amount of data indicates a role for the serotonergic system in this complex disorder. . Repeated cocaine use has been shown to increase DAT activity in humans (Mash et al., 2002). The ini- tial increase in extracellular dopamine after cocaine administration is thought to result in increased. et al., 2008). Interestingly, Chen and colleagues demonstrated that mutating S528 to alanine in DAT TMH 11 results in increased dopamine efflux (Chen and Justice, 2000). These find- ings suggest. GABA B receptor agonist, has resulted in increased cocaine abstinence in cocaine-addicted patients (Shoptaw et al., 2003). Similarly, the GABA transporter inhibitor tiagabine that is used for treating epilepsy has