In vivo analysis of serotonin clearance in rat hippocampus reveals that repeated administration of p-methoxyamphetamine (PMA), but not 3,4-methylenedioxymethamphetamine (MDMA), leads to long-lasting deficits in serotonin transporter function

p-Methoxyamphetamine (PMA) has been implicated in fatalities as a result of 'ecstasy' (MDMA) overdose worldwide. Like MDMA, acute effects are associated with marked changes in serotonergic neurotransmission, but the long-term effects of PMA are poorly understood. The aim of this study was to determine the effect of repeated PMA administration on in vitro measures of neurodegeneration: serotonin (5-HT) uptake, 5-HT transporter (SERT) density and 5-HT content in the hippocampus, and compare with effects on in vivo 5-HT clearance. Male rats received PMA, MDMA (4 or 15 mg/kg s.c., twice daily) or vehicle for 4 days and 2 weeks later indices of SERT function were measured. [(3)H]5-HT uptake into synaptosomes and [(3)H]cyanoimipramine binding to the SERT were significantly reduced by both PMA and MDMA treatments. 5-HT content was reduced in MDMA-, but not PMA-treatment. In contrast, clearance of locally applied 5-HT measured in vivo by chronoamperometry was only reduced in rats treated with 15 mg/kg PMA. The finding that 5-HT clearance in vivo was unaltered by MDMA treatment suggests that in vitro measures of 5-HT axonal degeneration do not necessarily predict potential compensatory mechanisms that maintain SERT function under basal conditions.     

In Vivo Electrochemical Studies of Dopamine Clearance in the Rat Substantia Nigra: Effects of Locally Applied Uptake Inhibitors and Unilateral 6-Hydroxydopamine Lesions

Abstract: High-speed chronoamperometric recordings were used to measure the uptake and clearance of locally applied dopamine (DA) within the substantia nigra (SN) of anesthetized rats. To establish that DA clearance within the SN was mediated primarily by the DA transporter (DAT) rather than the norepinephrine transporter (NET) or the serotonin transporter (SERT), we locally applied uptake inhibitors with different selectivity profiles for the various amine transporters. Nomifensine, a DAT/NET inhibitor, significantly potentiated both the amplitude and the time course of the DA signals. In contrast, neither the selective NET inhibitor desipramine, nor the selective SERT inhibitor citalopram affected the DA signal, suggesting that NET and SERT do not contribute to DA uptake and clearance within the regions of the SN studied over the concentration ranges (1–5 µ M ) used. In unilaterally 6-hydroxydopamine-lesioned rats, the time course of the DA signal was increased in both the lesioned SN and striatum, relative to the unlesioned hemisphere, indicating loss of DAT and decreased DA uptake and clearance. In addition, when identical amounts of DA were injected in the striatum and SN, peak signal amplitudes were larger in the SN, suggesting that the amplitudes are related to the number of DAT sites in a given region of brain tissue. For signals of equivalent amplitudes, clearance rates were lower in the SN than in the striatum, consistent with a lower capacity for DAT-mediated DA uptake within the SN. These results suggest that the DAT is the major transporter responsible for DA clearance within the rat SN.     

Serotonin (5-HT) transport in human platelets is modulated by Src-catalysed Tyr-phosphorylation of the plasma membrane transporter SERT.

BACKGROUND/AIM: platelets possess tightly regulated systems for serotonin (5-HT) transport. This study analysed whether the 5-HT transport mediated by the plasma-membrane transporter SERT is regulated by its Tyr-phosphorylation. METHODS: 5-HT transport was determined by filtration techniques, while immunoblotting procedures were adopted for detecting the Tyr-phosphorylation of SERT in human platelet fractions. RESULTS: 5-HT accumulation in platelets pre-treated with reserpine, which prevents the neurotransmitter transport into the dense granules, decreased upon cellular exposure to PP2 and SU6656, two structurally unrelated inhibitors of Src-kinases. By contrast, the protein Tyr-phosphatase inhibitor pervanadate increased the 5-HT accumulation. Anti-SERT immunostaining of the platelet fractions showed a major band displaying an apparent molecular mass of 50 kappaDa, indicating that, during the analytical procedure, SERT underwent proteolysis, which was counteracted by addition of 4 M urea in the cellular disrupting medium. The Tyr-phosphorylation degree of SERT immunoprecipitated from membrane extracts decreased by platelet treatment with SU6656 or PP2, and enhanced upon pervanadate treatment. The anti-SERT immunoprecipitates displayed anti-Src immunostaining and in vitro kinase activity towards a Src-specific peptide-substrate. Platelet treatment with PP2 or SU6656 also caused a decrease in the imipramine binding to platelets. It was concluded that the Src-mediated SERT Tyr-phosphorylation regulates the 5-HT transport by affecting the neurotransmitter binding sites.     

The cytoplasmic substrate permeation pathway of serotonin transporter

Serotonin transporter (SERT) catalyzes reuptake of the neurotransmitter serotonin (5-HT) and is a target for antidepressant drugs and psychostimulants. It is a member of a large family of neurotransmitter and amino acid transporters. A recent study using site-directed cysteine modification identified a helical region of the transporter with high accessibility to the cytoplasm. Subsequently, the high resolution structure of LeuT, a prokaryotic homologue, showed that the residues corresponding to this helical region are part of the fifth transmembrane domain. The accessibility of these positions is now shown to depend on conformational changes corresponding to interconversion of SERT between two forms that face the extracellular medium and the cytoplasm, respectively. Binding of the extracellular inhibitor cocaine decreased accessibility at these positions, whereas 5-HT, the transported substrate, increased it. The effect of 5-HT required the simultaneous presence of Na+ and Cl-, which are transported into the cell together (symported) with 5-HT. In light of the LeuT structure, these results begin to define the pathway through which 5-HT diffuses between its binding site and the cytoplasm. They also confirm a prediction of the alternating access model for transport, namely, that all symported substrates must bind together before translocation.     

Enhancement of 3,4-methylenedioxymethamphetamine neurotoxicity by the energy inhibitor malonate

The acute and long-term effects of the local perfusion of 3,4-methylenedioxymethamphetamine (MDMA) and the interaction with the mitochondrial inhibitor malonate (MAL) were examined in the rat striatum. MDMA, MAL or the combination of MAL with MDMA was reverse dialyzed into the striatum for 8 h via a microdialysis probe while extracellular dopamine (DA) and serotonin (5-HT) were measured. One week later, tissue immediately surrounding the probe was assayed for DA and 5-HT tissue content. Local perfusion of MDMA increased DA and 5-HT release but did not produce long-term depletion of DA or 5-HT in tissue. Malonate also increased both DA and 5-HT release but, in contrast to MDMA, produced only long-term depletion of DA. The combined perfusion of MDMA/MAL synergistically increased the release of DA and 5-HT and produced long-term depletion of both DA and 5-HT in tissue. These results support the conclusion that DA, compared with 5-HT, neurons are more susceptible to mitochondrial inhibition. Moreover, MDMA, which does not normally produce DA depletion in the rat, exacerbated MAL-induced DA depletions. The effect of MDMA in combination with MAL to produce 5-HT depletion suggests a role for bio-energetic stress in MDMA-induced toxicity to 5-HT neurons. Overall, these results highlight the importance of energy balance to the function of DA and 5-HT neurons and to the toxic effects of MDMA.     

Duloxetine inhibits effects of MDMA ("ecstasy") in vitro and in humans in a randomized placebo-controlled laboratory study

This study assessed the effects of the serotonin (5-HT) and norepinephrine (NE) transporter inhibitor duloxetine on the effects of 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) in vitro and in 16 healthy subjects. The clinical study used a double-blind, randomized, placebo-controlled, four-session, crossover design. In vitro, duloxetine blocked the release of both 5-HT and NE by MDMA or by its metabolite 3,4-methylenedioxyamphetamine from transmitter-loaded human cells expressing the 5-HT or NE transporter. In humans, duloxetine inhibited the effects of MDMA including elevations in circulating NE, increases in blood pressure and heart rate, and the subjective drug effects. Duloxetine inhibited the pharmacodynamic response to MDMA despite an increase in duloxetine-associated elevations in plasma MDMA levels. The findings confirm the important role of MDMA-induced 5-HT and NE release in the psychotropic effects of MDMA. Duloxetine may be useful in the treatment of psychostimulant dependence. TRIAL REGISTRATION: Clinicaltrials.gov NCT00990067.     

Effect of 3,4-Methylenedioxymethamphetamine on 3,4-Dihydroxyphenylalanine Accumulation in the Striatum and Nucleus Accumbens

The effect of the racemic mixture of 3,4-methylenedioxymethamphetamine (MDMA) on the synthesis of dopamine in the terminals of nigrostriatal and mesolimbic neurons was estimated by measuring the accumulation of 3,4-dihydroxyphenylalanine (DOPA) in the striatum and nucleus accumbens 30 min following the administration of the L-aromatic amino acid decarboxylase inhibitor, 3-hydroxybenzylhydrazine. MDMA produced an increase in DOPA accumulation in the striatum which was greater in magnitude and longer in duration than that in the nucleus accumbens. Although the concentrations of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in both the striatum and nucleus accumbens were reduced 3 h following an injection of MDMA (20 mg/kg), 5-HT and 5-HIAA concentrations were significantly reduced only in the striatum 7 days after the administration of MDMA. Pretreatment with a 5-HT2 antagonist, ketanserin, significantly attenuated the reduction in 5-HT concentration in the striatum 3 h following MDMA administration and completely blocked 5-HT depletion at 7 days post administration. Moreover, ketanserin completely blocked MDMA-induced DOPA accumulation in the striatum. The results obtained in these studies suggest that MDMA activates nigrostriatal dopaminergic pathways via 5-HT2 receptors. In addition, these data are supportive of the hypothesis that dopamine plays a role in MDMA-induced 5-HT depletion.     

Dopamine Transporter Loss in 6-OHDA Parkinson’s Model Is Unmet by Parallel Reduction in Dopamine Uptake

The dopamine transporter (DAT) regulates synaptic dopamine (DA) in striatum and modulation of DAT can affect locomotor activity. Thus, in Parkinson’s disease (PD), DAT loss could affect DA clearance and locomotor activity. The locomotor benefits of L-DOPA may be mediated by transport through monoamine transporters and conversion to DA. However, its impact upon DA reuptake is unknown and may modulate synaptic DA. Using the unilateral 6-OHDA rat PD model, we examined [³H]DA uptake dynamics in relation to striatal DAT and tyrosine hydroxylase (TH) protein loss compared with contralateral intact striatum. Despite >70% striatal DAT loss, DA uptake decreased only ∼25% and increased as DAT loss approached 99%. As other monoamine transporters can transport DA, we determined if norepinephrine (NE) and serotonin (5-HT) differentially modulated DA uptake in lesioned striatum. Unlabeled DA, NE, and 5-HT were used, at a concentration that differentially inhibited DA uptake in intact striatum, to compete against [³H]DA uptake. In 6-OHDA lesioned striatum, DA was less effective, whereas NE was more effective, at inhibiting [³H]DA uptake. Furthermore, norepinephrine transporter (NET) protein levels increased and desipramine was ∼two-fold more effective at inhibiting NE uptake. Serotonin inhibited [³H]DA uptake, but without significant difference between lesioned and contralateral striatum. L-DOPA inhibited [³H]DA uptake two-fold more in lesioned striatum and inhibited NE uptake ∼five-fold more than DA uptake in naïve striatum. Consequently, DA uptake may be mediated by NET when DAT loss is at PD levels. Increased inhibition of DA uptake by L-DOPA and its preferential inhibition of NE over DA uptake, indicates that NET-mediated DA uptake may be modulated by L-DOPA when DAT loss exceeds 70%. These results indicate a novel mechanism for DA uptake during PD progression and provide new insight into how L-DOPA affects DA uptake, revealing possible mechanisms of its therapeutic and side effect potential.     

MDMA神经毒性长期效应导致皮层和海马组织结构改变

通过短时间多次给药建立3,4．亚甲基二氧基甲基苯丙胺（3,4-methylenedioxymethamphetamine,MDMA）的神经毒性模型,将雄性Wistar大鼠随机分为对照组和实验组,实验组给予MDMA10mg／kg,每小时一次,共4次,即总量为40mg／kg,对照组给予等体积生理盐水。于末次给药后32周采用原位杂交检测5-HT转运体（serotonin transporter,SERT）mRNA和内源性焦虑物质苯甲二氮革结合性抑制物（diazepam binding inhibitor,DBI）的mRNA表达,免疫组织化学染色检测胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）的表达,银染观察神经末梢变化。结果显示,短时间多次给予MDMA后,与生理盐水组比较,MDMA组大鼠海马SERTmRNA信号表达降低（P〈0．05）,大脑皮层DBImRNA的信号表达增高（P〈0．05）,GFAP表达显著升高（P〈0．05）;银染MDMA组大鼠皮层神经末梢明显减少。上述结果提示,MDMA神经毒性导致皮层和海马结构改变持续存在,进而导致脑功能的紊乱。     

Cocaine Modulates Locomotion Behavior in C. elegans

Cocaine, a potent addictive substance, is an inhibitor of monoamine transporters, including DAT (dopamine transporter), SERT (serotonin transporter) and NET (norepinephrine transporter). Cocaine administration induces complex behavioral alterations in mammals, but the underlying mechanisms are not well understood. Here, we tested the effect of cocaine on C. elegans behavior. We show for the first time that acute cocaine treatment evokes changes in C. elegans locomotor activity. Interestingly, the neurotransmitter serotonin, rather than dopamine, is required for cocaine response in C. elegans. The C. elegans SERT MOD-5 is essential for the effect of cocaine, consistent with the role of cocaine in targeting monoamine transporters. We further show that the behavioral response to cocaine is primarily mediated by the ionotropic serotonin receptor MOD-1. Thus, cocaine modulates locomotion behavior in C. elegans primarily by impinging on its serotoninergic system.     

Ketanserin pretreatment attenuates MDMA-induced dopamine release in the striatum as measured by in vivo microdialysis

Systemic administration of the amphetamine analogue, 3,4-methylenedioxymethamphetamine (MDMA) produced a dose-dependent increase in the extracellular concentration of dopamine (DA) in the striatum as measured by in vivo microdialysis in awake, freely-moving rats. The extracellular concentration of the DA metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), was significantly decreased in dialysate samples following the administration of MDMA (10 and 20 mg/kg, i.p.). The serotonin-2 (5-HT2) antagonist ketanserin (3 mg/kg, i.p.) had no effect on the extracellular concentration of DA or DOPAC in the striatum of vehicle- treated rats. The administration of ketanserin (3 mg/kg) 1 hr prior to MDMA (20 mg/kg) significantly attenuated the MDMA- induced increase in the extracellular concentration of DA without affecting the decrease in DOPAC concentrations. These data are suggestive that MDMA administration increases DA release in the striatum of awake, freely-moving rats. In addition, MDMA-induced increase in the extracellular concentration of DA in the striatum is mediated, in part, via 5-HT2 receptor mechanisms.     

Expression and function of 5-HT3 receptors in the enteric neurons of mice lacking the serotonin transporter

The actions of enteric 5-HT are terminated by 5-HT transporter (SERT)-mediated uptake, and gastrointestinal motility is abnormal in SERT -/- mice. We tested the hypothesis that adaptive changes in enteric 5-HT(3) receptors help SERT -/- mice survive despite inefficient 5-HT inactivation. Expression of mRNA encoding enteric 5-HT(3A) subunits was similar in SERT +/+ and -/- mice, but that of 5-HT(3B) subunits was fourfold less in SERT -/- mice. 5-HT(3B) mRNA was found, by in situ hybridization, in epithelial cells and enteric neurons. 5-HT evoked a fast inward current in myenteric neurons that was pharmacologically identified as 5-HT(3) mediated. The EC(50) of the 5-HT response was lower in SERT +/+ (18 microM) than in SERT -/- (36 microM) mice and desensitized rapidly in a greater proportion of SERT -/- neurons; however, peak amplitudes, steady-state current, and decay time constants were not different. Adaptive changes thus occur in the subunit composition of enteric 5-HT(3) receptors of SERT -/- mice that are reflected in 5-HT(3) receptor affinity and desensitization.     

Carrier-Mediated Release of Serotonin by 3,4-Methylenedioxymethamphetamine: Implications for Serotonin-Dopamine Interactions

Abstract: In vivo microdialysis was used to determine whether the 3,4-methylenedioxymethamphetamine (MDMA)-induced release of serotonin (5-HT) in vivo involves a carrier-mediated process and to investigate further the state-dependent interaction between 5-HT and dopamine. MDMA produced a dose-dependent increase in the extracellular concentration of 5-HT in the striatum and prefrontal cortex that was attenuated by treatment with fluoxetine but not by tetrodotoxin. Suppression by fluoxetine of the MDMA-induced release of 5-HT was accompanied by a suppression of the MDMA-induced release of dopamine. Administration of MDMA to rats treated with carbidopa and l -5-hydroxytryptophan resulted in a synergistic elevation of the extracellular concentration of 5-HT that was much greater than that produced by either treatment alone. The MDMA-induced release of dopamine by MDMA also was potentiated in 5-hydroxytryptophan-treated rats. These data are consistent with the view that MDMA increases the extracellular concentration of 5-HT by facilitating carrier-mediated 5-HT release, which can be enhanced greatly under conditions in which 5-HT synthesis is stimulated. Moreover, these data are supportive of a state-dependent, stimulatory role of 5-HT in the regulation of dopamine release.     

Further structural optimization of cis-(6-benzhydryl-piperidin-3-yl)-benzylamine and 1,4-diazabicyclo[3.3.1]nonane derivatives by introducing an exocyclic hydroxyl group: interaction with dopamine, serotonin, and norepinephrine transporters

Our earlier effort to develop constrained analogues of flexible piperidine derivatives for monoamine transporters led to the development of a series of 3,6-disubstituted piperidine derivatives, and a series of 4,8-disubstituted 1,4-diazabicyclo[3.3.1]nonane derivatives. In further structure-activity relationship (SAR) studies on these constrained derivatives, several novel analogues were developed where an exocyclic hydroxyl group was introduced on the N-alkyl-aryl side chain. All synthesized derivatives were tested for their affinities for the dopamine transporter (DAT), serotonin (5-HT) transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting the uptake of [(3)H]DA, [(3)H]5-HT, and [(3)H]NE, respectively. Compounds were also tested for their binding potency at the DAT by their ability to inhibit binding of [(3)H]WIN 35,428. The results indicated that position of the hydroxyl group on the N-alkyl side chain is important along with the length of the side chain. In general, hydroxyl derivatives derived from more constrained bicyclic diamines exhibited greater selectivity for interaction with DAT compared to the corresponding 3,6-disubstituted diamines. In the current series of molecules, compound 11b with N-propyl side chain with the hydroxyl group attached in the benzylic position was the most potent and selective for DAT (K(i)=8.63nM; SERT/DAT=172 and NET/DAT=48.4).     

The role of dopamine and serotonin in regulating bone mass and strength: studies on dopamine and serotonin transporter null mice

Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. Dopamine (DA) has been reported to have effects on calcium and phosphorus metabolism. The dopamine transporter (DAT) is believed to control the temporal and spatial activity of released DA by rapid uptake of the neurotransmitter into presynaptic terminals. We have evaluated the histologic and biomechanical properties of the skeleton in mice homozygous for deletion of the DA transporter gene (DAT (-/-)) to help delineate the role of DA in bone biology. We have demonstrated that DAT (-/-) mice have reduced bone mass and strength. DAT (-/-) animals have shorter femur length and dry weight, and lower ash calcium content. Cancellous bone volume in the DAT (-/-) proximal tibial metaphysis is significantly decreased with reduced trabecular thickness. DAT (-/-) vertebrae have lower cancellous bone volume as a consequence of increased trabecular spacing and reduced trabecular number, and cortical thickness and bone area in the femoral diaphysis are reduced. The ultimate bending load (femoral strength) for the DAT (-/-) mice is 30% lower than the wild-type mice. Thus, deletion of the DAT gene results in deficiencies in skeletal structure and integrity. Since serotonin (5-HT) plays a role as a regulator of craniofacial morphogenesis, we explored the expression and function of 5-HT receptors and the 5-HT transporter (5-HTT) in bone. Primary cultures of rat osteoblasts (rOB) and a variety of clonal osteoblastic cell lines including ROS 17/2.8, UMR 106-H5 and Py1a show mRNA expression for the 5-HTT, and the 5-HT(1A), 5-HT(1D), 5-HT(2A) and 5-HT(2B) receptors by RT-PCR analysis and immunoblot. A relatively high density of nanomolar affinity 5-HTT binding sites is present in ROS 17/2.8 and UMR 106-H5 cells. The maximal [(3)H]5-HT uptake rate in ROS cells was 110 pmol/10 min/well, with a K(m) value of 1.13 microM. In normal differentiating rOB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased by almost eight-fold at day 31. In mature rOB cultures, the estimated density of [(125)I]RTI-55 binding sites was 600 fmol/mg protein. PMA treatment caused a significant 40% reduction in the maximal uptake rate of [(3)H]5-HT, an effect prevented by pretreatment with staurosporine. 5-HT potentiates the PTH-induced increase in AP-1 activity in UMR 106-H5 cells. In 5-HTT (-/-) animals, cancellous bone volume (BV/TV) in the lumbar vertebrae is reduced, with a trend toward decreased trabecular thickness and trabecular number. These results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT, and disruption of the 5-HTT gene may cause osteopenia.     

Mazindol Attenuates the 3,4-Methylenedioxymethamphetamine-Induced Formation of Hydroxyl Radicals and Long-Term Depletion of Serotonin in the Striatum

The formation of hydroxyl radicals following the systemic administration of 3,4-methylenedioxymethamphetamine (MDMA) was studied in the striatum of the rat by quantifying the stable adducts of salicylic acid and D-phenylalanine, namely, 2,3-dihydroxybenzoic acid (2,3-DHBA) and p-tyrosine, respectively. The repeated administration of MDMA produced a sustained increase in the extracellular concentration of 2,3-DHBA and p-tyrosine, as well as dopamine. The MDMA-induced increase in the extracellular concentration of both dopamine and 2,3-DHBA was suppressed in rats treated with mazindol, a dopamine uptake inhibitor. Mazindol also attenuated the long-term depletion of serotonin (5-HT) in the striatum produced by MDMA without altering the acute hyperthermic response to MDMA. These results are supportive of the view that MDMA produces a dopamine-dependent increase in the formation of hydroxyl radicals in the striatum that may contribute to the mechanism whereby MDMA produces a long-term depletion of brain 5-HT content.     

Nonexocytotic serotonin release tonically suppresses serotonergic neuron activity

The firing activity of serotonergic neurons in raphe nuclei is regulated by negative feedback exerted by extracellular serotonin (5-HT)_o acting through somatodendritic 5-HT1A autoreceptors. The steady-state [5-HT]_o, sensed by 5-HT1A autoreceptors, is determined by the balance between the rates of 5-HT release and reuptake. Although it is well established that reuptake of 5-HT_o is mediated by 5-HT transporters (SERT), the release mechanism has remained unclear. It is also unclear how selective 5-HT reuptake inhibitor (SSRI) antidepressants increase the [5-HT]_o in raphe nuclei and suppress serotonergic neuron activity, thereby potentially diminishing their own therapeutic effect. Using an electrophysiological approach in a slice preparation, we show that, in the dorsal raphe nucleus (DRN), continuous nonexocytotic 5-HT release is responsible for suppression of phenylephrine-facilitated serotonergic neuron firing under basal conditions as well as for autoinhibition induced by SSRI application. By using 5-HT1A autoreceptor-activated G protein–gated inwardly rectifying potassium channels of patched serotonergic neurons as 5-HT_o sensors, we show substantial nonexocytotic 5-HT release under conditions of abolished firing activity, Ca²⁺ influx, vesicular monoamine transporter 2–mediated vesicular accumulation of 5-HT, and SERT-mediated 5-HT transport. Our results reveal a cytosolic origin of 5-HT_o in the DRN and suggest that 5-HT_o may be supplied by simple diffusion across the plasma membrane, primarily from the dense network of neurites of serotonergic neurons surrounding the cell bodies. These findings indicate that the serotonergic system does not function as a sum of independently acting neurons but as a highly interdependent neuronal network, characterized by a shared neurotransmitter pool and the regulation of firing activity by an interneuronal, yet activity-independent, nonexocytotic mechanism.     

Brain serotonin transporter binding of [123I]ADAM: within-subject variation between summer and winter data

The neurotransmitter serotonin (5-HT) controls several physiological functions, and a disturbance of the 5-HT system is implicated in many psychiatric conditions. Seasonal variation has been suggested in the 5-HT system. We investigated within-subject seasonal variation in brain serotonin transporter (SERT) binding with the SERT-ligand [(123)I]ADAM and single photon emission computed tomography (SPECT) in 12 healthy individuals. No systematic variation was found in the midbrain or thalamus areas between scans done in summer and winter. Our results suggest that factors other than season are more important in causing within-subject variation of brain SERT binding between summer and winter.     

Serotonin (5-HT) release and uptake measured by real-time electrochemical techniques in the rat ileum

Serotonin (5-HT) is released from the enterochromaffin cells and plays an important role in regulating intestinal function. Although the release of 5-HT is well documented, the contribution of the serotonin reuptake transporter (SERT) to the levels and actions of 5-HT in the intestine is unclear. This study aimed to demonstrate real-time SERT activity in ileal mucosa and to assess the effects of SERT inhibition using fluoxetine. Electrochemical recordings were made from the mucosa in full-thickness preparations of rat ileum using a carbon fiber electrode to measure 5-HT oxidation current and a force transducer to record circular muscle (CM) tension. Compression of the mucosa stimulated a peak 5-HT release of 12 +/- 6 microM, which decayed to 7 +/- 4 microM. Blockade of SERT with fluoxetine (1 microM) increased the peak compression-evoked release to 19 +/- 9 microM, and the background levels of 5-HT increased to 11 +/- 7 microM (P < 0.05, n = 7). When 5-HT was exogenously applied to the mucosa, fluoxetine caused a significant increase in the time to 50% and 80% decay of the oxidation current. Fluoxetine also increased the spontaneous CM motility (P < 0.05; n = 7) but did not increase the CM contraction-evoked 5-HT release (P > 0.05, n = 5). In conclusion, this is the first characterization of the real-time uptake of 5-HT into the rat intestine. These data suggest that SERT plays an important role in the modulation of 5-HT concentrations that reach intestinal 5-HT receptors.     

5-HT(1B) receptor-mediated regulation of serotonin clearance in rat hippocampus in vivo

The 5-hydroxytryptamine (5-HT; serotonin) transporter (5-HTT) is important in terminating serotonergic neurotransmission and is a primary target for many psychotherapeutic drugs. Study of the regulation of 5-HTT activity is therefore important in understanding the control of serotonergic neurotransmission. Using high-speed chronoamperometry, we have demonstrated that local application of 5-HT(1B) antagonists into the CA3 region of the hippocampus prolongs the clearance of 5-HT from extracellular fluid (ECF). In the present study, we demonstrate that the 5-HT(1B) antagonist cyanopindolol does not produce this effect by increasing release of endogenous 5-HT or by directly binding to the 5-HTT. Dose-response studies showed that the potency of cyanopindolol to inhibit clearance of 5-HT was equivalent to that of the selective 5-HT reuptake inhibitor fluvoxamine. Local application of the 5-HT(1A) antagonist WAY 100635 did not alter 5-HT clearance, suggesting that the effect of cyanopindolol to prolong clearance is not via a mechanism involving 5-HT(1A) receptors. Finally, the effect of low doses of cyanopindolol and fluvoxamine to inhibit clearance of 5-HT from ECF was additive. These data are consistent with the hypothesis that activation of terminal 5-HT(1B) autoreceptors increases 5-HTT activity.