Serotonin and epilepsy

In recent years, there has been increasing evidence that serotonergic neurotransmission modulates a wide variety of experimentally induced seizures. Generally, agents that elevate extracellular serotonin (5-HT) levels, such as 5-hydroxytryptophan and serotonin reuptake blockers, inhibit both focal and generalized seizures, although exceptions have been described, too. Conversely, depletion of brain 5-HT lowers the threshold to audiogenically, chemically and electrically evoked convulsions. Furthermore, it has been shown that several anti-epileptic drugs increase endogenous extracellular 5-HT concentration. 5-HT receptors are expressed in almost all networks involved in epilepsies. Currently, the role of at least 5-HT(1A), 5-HT(2C), 5-HT(3) and 5-HT(7) receptor subtypes in epileptogenesis and/or propagation has been described. Mutant mice lacking 5-HT(1A) or 5-HT(2C) receptors show increased seizure activity and/or lower threshold. In general, hyperpolarization of glutamatergic neurons by 5-HT(1A) receptors and depolarization of GABAergic neurons by 5-HT(2C) receptors as well as antagonists of 5-HT(3) and 5-HT(7) receptors decrease the excitability in most, but not all, networks involved in epilepsies. Imaging data and analysis of resected tissue of epileptic patients, and studies in animal models all provide evidence that endogenous 5-HT, the activity of its receptors, and pharmaceuticals with serotonin agonist and/or antagonist properties play a significant role in the pathogenesis of epilepsies.     

Serotonin and fluoxetine modulate bone cell function in vitro

Recent studies have proposed a role for serotonin and its transporter in regulation of bone cell function. In the present study, we examined the in vitro effects of serotonin and the serotonin transporter inhibitor fluoxetine "Prozac" on osteoblasts and osteoclasts. Human mononuclear cells were differentiated into osteoclasts in the presence of serotonin or fluoxetine. Both compounds affected the total number of differentiated osteoclasts as well as bone resorption in a bell-shaped manner. RT-PCR on the human osteoclasts demonstrated several serotonin receptors, the serotonin transporter, and the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase 1 (Tph1). Tph1 expression was also found in murine osteoblasts and osteoclasts, indicating an ability to produce serotonin. In murine pre-osteoclasts (RAW264.7), serotonin as well as fluoxetine affected proliferation and NFkappaB activity in a biphasic manner. Proliferation of human mesenchymal stem cells (MSC) and primary osteoblasts (NHO), and 5-HT2A receptor expression was enhanced by serotonin. Fluoxetine stimulated proliferation of MSC and murine preosteoblasts (MC3T3-E1) in nM concentrations, microM concentrations were inhibitory. The effect of fluoxetine seemed direct, probably through 5-HT2 receptors. Serotonin-induced proliferation of MC3T3-E1 cells was inhibited by the PKC inhibitor (GF109203) and was also markedly reduced when antagonists of the serotonin receptors 5-HT2B/C or 5-HT2A/C were added. Serotonin increased osteoprotegerin (OPG) and decreased receptor activator of NF-kappaB ligand (RANKL) secretion from osteoblasts, suggesting a role in osteoblast-induced inhibition of osteoclast differentiation, whereas fluoxetine had the opposite effect. This study further describes possible mechanisms by which serotonin and the serotonin transporter can affect bone cell function.     

Serotonin decreases generation of dopaminergic neurons from mesencephalic precursors via serotonin type 7 and type 4 receptors

Inductive signals mediating the differentiation of neural precursors into serotonergic (5-HT) or dopaminergic neurons have not been clarified. We have recently shown that in cell aggregates obtained from rat mesencephalic precursors, reduction of serotonin levels induces a marked increase in generation of dopaminergic neurons. In the present study we treated rat neurospheres with antagonists of the main subtypes of 5-HT receptors, 5-HT transport inhibitors, or 5-HT receptor agonists, and studied the effects on generation of dopaminergic neurons. Cultures treated with Methiothepin (5-HT(1,2,5,6,7) receptor antagonist), the 5-HT(4) receptor antagonist GR113808;67:00-.or the 5-HT(7) receptor antagonist SB 269970 showed a significant increase in generation of dopaminergic cells. Treatment with the 5-HT(1B/1D) antagonist GR 127935, the 5-HT(2) antagonist Ritanserin, the 5-HT transporter inhibitor Fluoxetine, the dopamine and norepinephrine transport inhibitor GBR 12935, or with both inhibitors together, or 5-HT(4) or 5-HT(7) receptor agonists induced significant decreases in generation of dopaminergic cells. Cultures treated with WAY100635 (5-HT(1A) receptor antagonist), the 5-HT(3) receptor antagonist Ondasetron, or the 5-HT(6) receptor antagonist SB 258585 did not show any significant changes. Therefore, 5-HT(4) and 5-HT(7) receptors are involved in the observed serotonin-induced decrease in generation of dopaminergic neurons from proliferating neurospheres of mesencephalic precursors. 5-HT(4) and 5-HT(7) receptors were found in astrocytes and serotonergic cells using double immunolabeling and laser confocal microscopy, and the glial receptors appeared to play a major role.     

Mutagenesis analysis of the serotonin 5-HT2C receptor and a Caenorhabditis elegans 5-HT2 homologue: conserved residues of helix 4 and helix 7 contribute to agonist-dependent activation of 5-HT2 receptors

An alignment of serotonin [5-hydroxytryptamine (5-HT)] G protein-coupled receptors identified a lysine at position 4.45 (helix 4) and a small polar residue (serine or cysteine) at 7.45 (helix 7) that occur exclusively in the 5-HT2 receptor family. Other serotonin receptors have a hydrophobic amino acid, typically a methionine, at 4.45 and an invariant asparagine at 7.45. The functional significance of these class-specific substitutions was tested by site-directed mutagenesis of two distantly related 5-HT2 receptors, Caenorhabditis elegans 5-HT2ce and rat 5-HT2C. Residues 4.45 and 7.45 were each mutated to a methionine and asparagine, respectively, or an alanine and the resulting constructs were tested for activity. A K4.45M mutation decreased serotonin-dependent activity (Emax) of the rat 5-HT2C receptor by 60% and that of the C. elegans homologue by 40%, as determined by a fluorometric plate-based calcium assay. The rat mutant also exhibited nearly sixfold higher agonist binding affinity and significantly lower constitutive activity compared with wildtype. Mutagenesis of S7.45 in the C. elegans receptor increased serotonin binding affinity by up to 25-fold and decreased Emax by up to 65%. The same mutations of the cognate C7.45 in rat 5-HT2C produced a smaller fourfold change in the affinity for serotonin and decreased agonist efficacy by up to 50%. Substitutions of S/C7.45 did not produce a significant change in the basal activity of either receptor. All mutants tested exhibited levels of receptor expression similar to the corresponding wildtype based on measurements of specific [3H]-mesulergine binding or flow cytometry analyses. Taken together, these results suggest that K4.45 and S/C7.45 play an important role in the conformational rearrangements leading to agonist-induced activation of 5-HT2 receptors.     

Serotonin Regulation of Serotonin Uptake in RN46A Cells

1. Aim: The role of the serotonin transporter (SERT) is to remove serotonin (5-HT) from the synaptic space. In vitro studies have shown that 5-HT uptake via SERT is influenced by the availability of its substrate, 5-HT. We used RN46A cells, a line that expresses SERT, to investigate 5-HT regulation of 5-HT uptake and the intracellular signaling pathways involved. RN46A cells also express mRNAs for 5-HT receptors (5-HT_1A, 5-HT_1B, 5-HT_2A, and 5-HT_2C) and as cAMP and intracellular Ca²⁺ are modulated by different 5-HT receptors, we studied both pathways.2. Methods: 5-HT uptake was determined as imipramine-inhibitable uptake of [³H]5-HT, intracellular cAMP was measured by RIA and intracellular Ca²⁺ changes were determined using the ratiometric method of intracellular Ca²⁺ imaging.3. Results: For uptake experiments, cells were kept for 30 min either with or without 1 μM 5-HT in the medium before measuring uptake. Removal of 5-HT for 30 min significantly decreased [³H]5-HT uptake. The absence of 5-HT for 15 min failed to induce any changes in intracellular cAMP levels. Removal of 5-HT from the medium did not change intracellular Ca²⁺ levels either; however, adding 1 μM 5-HT after 5 min in 5-HT-free conditions rapidly increased intracellular Ca²⁺ levels in 50% of the cells. The remaining cells showed no changes in the intracellular Ca²⁺ levels.4. Conclusions: We have shown that in RN46A cells, that endogenously express SERT and mRNAs for several 5-HT receptors, changes in 5-HT levels influence 5-HT uptake rate as well as induce changes in intracellular Ca²⁺ levels. This suggests that 5-HT may utilize intracellular Ca²⁺ to regulate 5-HT uptake.     

The effects of serotonin agonists on the hypothalamic regulation of sexual receptivity in Syrian hamsters

One brain region that has been implicated in the regulation of lordosis is the medial preoptic-anterior hypothalamic continuum (MPOA-AH). Previous studies have suggested that this zone may be part of the circuit mediating the effects of serotonin (5-HT) on sexual receptivity. In the present experiments, we investigated the role of 5-HT(1a/7) and 5-HT(2) receptor subtypes in the MPOA-AH in the control of lordosis. In two experiments, either 5-HT(1a/7) or 5-HT(2) agonists were injected unilaterally into the MPOA-AH of ovariectomized, hormonally primed female hamsters. In the first experiment, microinjections of the 5-HT(1a/7) agonist 8-hydroxy-2,9-(di-n-propylamino)tetralin resulted in an attenuation of the lordosis posture by causing a decrease in the mean lordosis duration and an increase in the number of lordosis episodes over the entire testing period. In the second experiment, microinjections of the 5-HT(2b/2c) agonist m-chlorophenylpiperazine did not result in any changes in sexual receptivity. However, microinjections of the 5-HT(2) agonist (2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl facilitated lordosis by increasing the mean lordosis duration and decreasing the number of lordosis episodes in the first 5 min of the testing period. These data indicate that serotonin may act in the MPOA-AH via 5-HT(1a/7) receptors to attenuate and 5-HT(2) receptors to facilitate sexual receptivity.     

Effect of domoic acid on metabolism of 5-hydroxytryptamine in rat brain

Domoic acid (Dom) is a neurotoxic secondary amino acid that interacts with the glutamate receptors, producing neurological problems. In the present work, we study the effects of Dom on the levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in discrete rat brain regions. The effects of Dom on the brain metabolism of serotonin are also discussed in this paper. Dom stimulates the rat brain serotoninergic system, increasing differentially the synthesis and the catabolism of 5-HT and the elimination of 5-HIAA.     

Serotonin 5-HT(2A) receptor activation induces 2-arachidonoylglycerol release through a phospholipase c-dependent mechanism

To date, several studies have demonstrated that phospholipase C-coupled receptors stimulate the production of endocannabinoids, particularly 2-arachidonoylglycerol. There is now evidence that endocannabinoids are involved in phospholipase C-coupled serotonin 5-HT(2A) receptor-mediated behavioral effects in both rats and mice. The main objective of this study was to determine whether activation of the 5-HT(2A) receptor leads to the production and release of the endocannabinoid 2-arachidonoylglycerol. NIH3T3 cells stably expressing the rat 5-HT(2A) receptor were first incubated with [(3)H]-arachidonic acid for 24 h. Following stimulation with 10 mum serotonin, lipids were extracted from the assay medium, separated by thin layer chromatography, and analyzed by liquid scintillation counting. Our results indicate that 5-HT(2A) receptor activation stimulates the formation and release of 2-arachidonoylglycerol. The 5-HT(2A) receptor-dependent release of 2-arachidonoylglycerol was partially dependent on phosphatidylinositol-specific phospholipase C activation. Diacylglycerol produced downstream of 5-HT(2A) receptor-mediated phospholipase D or phosphatidylcholine-specific phospholipase C activation did not appear to contribute to 2-arachidonoylglycerol formation in NIH3T3-5HT(2A) cells. In conclusion, our results support a functional model where neuromodulatory neurotransmitters such as serotonin may act as regulators of endocannabinoid tone at excitatory synapses through the activation of phospholipase C-coupled G-protein coupled receptors.     

大鼠脑中5—羟色胺受体的检定及其在衰老时的变化

采用放射性配基结合分析法,对大鼠大脑皮质的5-HT受体作了检定,并观察了老年大鼠(36月龄)大脑皮质中该受体的变化。证实大鼠大脑皮质存在着丰富的、高亲和力和单一结合位点的5-HT受体。老年大鼠大脑皮质中5-HT受体的数目较成年大鼠(3月龄)明显减少,但亲和力无改变。应用荧光分光技术测定了成年和老年大鼠脑干和大脑皮质5-HT含量,证实老年大鼠上述两个脑区的5-HT含量均有降低。本研究的结果提示,老年大鼠中枢5-HT系统的功能减低,这一变化可能与老年期的一些表现如睡眠障碍、体温低、记忆力减退和易患精神疾病等有关。     

Quantitative analysis of glutamatergic and GABAergic neurons expressing 5-HT(2A) receptors in human and monkey prefrontal cortex

5-hydroxytryptamine (5-HT) or serotonin 2A receptors play an important role in modulation of prefrontal cortex (PFC) activity and have been implicated in the physiopathology of psychiatric disorders. There is no quantitative information on the percentage of glutamatergic and GABAergic cells that express 5-HT(2A) receptors in human and monkey PFC. We have used double in situ hybridization to quantify the mRNA co-localization of 5-HT(2A) receptor with the glutamatergic transporter vesicular glutamate transporter 1, and with the GABAergic marker glutamic acid decarboxylase 65/67 and in parvalbumin and calbindin GABAergic cell populations. Our results show that nearly every glutamatergic cell (86-100%) in layers II-V expressed 5-HT(2A) receptor mRNA in both species. This percentage was lower in layer VI (13-31%). In contrast, not all the GABAergic interneurons (13-46%) expressed 5-HT(2A) receptor mRNA. This receptor was expressed in 45-69% of parvalbumin and in 61-87% of calbindin positive cells. These results indicate that, while the majority of glutamatergic neurons can be sensitive to 5-HT action via 5-HT(2A) receptors, this modulation occurs only in a limited population of GABAergic interneurons and provides new neuroanatomical information about the role played by serotonin through 5-HT(2A) receptors in the PFC and on the sites of action for drugs such as antipsychotics and antidepressants used in treatment of psychiatric disorders.     

Exaggerated effect of fluvoxamine in heterozygote serotonin transporter knockout mice

Clearance rates for serotonin (5-HT) in heterozygote (+/-) and homozygote (-/-) serotonin transporter (5-HTT) knockout (KO) mice have not been determined in vivo. Moreover, the effect of selective serotonin reuptake inhibitors (SSRIs) on 5-HT clearance in these mice has not been examined. In this study, the rate of clearance of exogenously applied 5-HT was measured in the CA3 region of the hippocampus of anesthetized mice using high-speed chronoamperometry. Compared with wild-type mice, the maximal rate of 5-HT clearance from extracellular fluid (ECF) was decreased in heterozygotes and more markedly so in KO mice. Heterozygote mice were more sensitive to the 5-HT uptake inhibitor, fluvoxamine, resulting in longer clearance times for 5-HT than in wild-type mice; as expected, the KO mice were completely unresponsive to fluvoxamine. There were no associated changes in norepinephrine transporter density, nor was there an effect of the norepinephrine uptake inhibitor, desipramine, on 5-HT clearance in any genotype. Thus, adaptive changes in the norepinephrine transport system do not occur in the CA3 region of hippocampus as a consequence of 5-HTT KO. These data highlight the potential of the heterozygote 5-HTT mutant mice to model the dynamic in vivo consequences of the human 5-HTT polymorphism.     

5-HT(1A) receptor mutant mice exhibit enhanced tonic, stress-induced and fluoxetine-induced serotonergic neurotransmission

Mutant mice that lack serotonin(1A) receptors exhibit enhanced anxiety-related behaviors, a phenotype that is hypothesized to result from impaired autoinhibitory control of midbrain serotonergic neuronal firing. Here we examined the impact of serotonin(1A) receptor deletion on forebrain serotonin neurotransmission using in vivo microdialysis in the frontal cortex and ventral hippocampus of serotonin(1A) receptor mutant and wild-type mice. Baseline dialysate serotonin levels were significantly elevated in mutant animals as compared with wild-types both in frontal cortex (mutant = 0.44 +/- 0.05 n M; wild-type = 0.28 +/- 0.03 n M) and hippocampus (mutant = 0.46 +/- 0.07 n M; wild-type = 0.27 +/- 0.04 n M). A stressor known to elicit enhanced anxiety-like behaviors in serotonin(1A) receptor mutants increased dialysate 5-HT levels in the frontal cortex of mutant mice by 144% while producing no alteration in cortical 5-HT in wild-type mice. There was no phenotypic difference in the effect of this stressor on serotonin levels in the hippocampus. Fluoxetine produced significantly greater increases in dialysate 5-HT content in serotonin(1A) receptor mutants as compared with wild-types, with two- and three-fold greater responses being observed in the hippocampus and frontal cortex, respectively. This phenotypic effect was mimicked in wild-types by pretreatment with the serotonin(1A) antagonist 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide (p-MPPI). These results indicate that deletion of central serotonin(1A) receptors results in a tonic disinhibition of central serotonin neurotransmission, with a greater dysregulation of serotonin release in the frontal cortex than ventral hippocampus under conditions of stress or increased interstitial serotonin levels.     

Caveolin-1 affects serotonin binding and cell surface levels of human 5-HT7(a) receptors

Studies using HeLa cells expressing 5-HT7 receptors showed that detergent resistant membranous lipid rafts purified by sucrose gradient centrifugation contained 5-HT7 receptors and caveolin-1. Caveolin-1 siRNA-mediated knockdown or serotonin (5-HT) treatment caused significant reductions of maximum [3H]5-HT binding to 5-HT7 receptors and total immunoreactivity of membranous 5-HT7 receptors in intact cells. Co-treatment with 5-HT, caveolin-1 siRNA and methyl-beta-cyclodextrin had no additive effects on reducing maximum binding of [3H]5-HT to 5-HT7 receptors. 5-HT-mediated reduction of [3H]5-HT binding to 5-HT7 receptors was counteracted by genistein, but not by sucrose. Caveolin-1, specifically localized in cholesterol-enriched lipid rafts, appears to regulate constitutive and agonist-stimulated cell surface levels of 5-HT7 receptors via a clathrin-independent mechanism.     

Differential Effect of Subchronic Treatment with Various Neuroleptic Agents on Serotonin2 Receptors in Rat Cerebral Cortex

In addition to antidepressant drugs, some neuroleptic (NL) drugs reduce serotonin2 (5-HT2) receptor binding sites after chronic administration. The present study was undertaken to characterize further this property of NL drugs. Scatchard analysis of [3H]spiperone binding in rat cerebral cortex revealed that 21-day treatment with chlorpromazine (CPZ), cis-flupenthixol, and thioridazine reduced 5-HT2 radioligand binding density by 60, 27, and 18%, respectively. The more selective dopamine-D2 antagonists haloperidol and sulpiride were totally ineffective in this regard. No reduction in 5-HT2 ligand binding sites occurred after 1 day of treatment with CPZ but 3-days of treatment was effective and this reduction persisted, although diminished, for at least 72 h after the last injection. cis-Flupenthixol and d-butaclamol were also effective after 3 days of treatment but trans-flupenthixol and l-butaclamol were not, indicating stereo-specificity of the response mechanism. Female rats showed the same response to CPZ as did male rats. Central 5,7-dihydroxytryptamine-induced lesions of 5-HT neurons demonstrated that intact 5-HT neurons were not required for the reduction of 5-HT2 receptor ligand binding by CPZ. Since CPZ has high affinity for many receptors, including alpha 1, histamine1, and muscarinic receptors, the role of these effects in producing 5-HT2 receptor down-regulation was considered by studying the effects of prazosin, atropine, and pyrilamine administration on 5-HT2 radioligand binding. Results indicate that no one of these actions appears to account for the down-regulation of 5-HT2 receptors by CPZ. Several of these effects, in combination, or some unique mechanism, may be involved.     

Anatomical study of serotonergic innervation and 5-HT1A receptor in the human spinal cord

Serotonergic innervation of the spinal cord in mammals has multiple roles in the control of motor, sensory and visceral functions. In rats, functional consequences of spinal cord injury at thoracic level can be improved by a substitutive transplantation of serotonin (5-HT) neurons or regeneration under the trophic influence of grafted stem cells. Translation to either pharmacological and/or cellular therapies in humans requires the mapping of the spinal cord 5-HT innervation and its receptors to determine their involvement in specific functions. Here, we have performed a preliminary mapping of serotonergic processes and serotonin-lA (5-HT_1A) receptors in thoracic and lumbar segments of the human spinal cord. As in rodents and non-human primates, 5-HT profiles in human spinal cord are present in the ventral horn, surrounding motoneurons, and also contact their presumptive dendrites at lumbar level. 5-HT_1A receptors are present in the same area, but are more densely expressed at lumbar level. 5-HT profiles are also present in the intermediolateral region, where 5-HT_1A receptors are absent. Finally, we observed numerous serotonergic profiles in the superficial part (equivalent of Rexed lamina II) of the dorsal horn, which also displayed high levels of 5-HT_1A receptors. These findings pave the way for local specific therapies involving cellular and/or pharmacological tools targeting the serotonergic system.     

Differences in serotonergic neurotransmission between rats displaying high or low anxiety/depression-like behaviour: effects of chronic paroxetine treatment

Disturbances in serotonergic neurotransmission have been suggested to be closely interlinked with hyperactivity of the hypothalamic-pituitary-adrenocortical (HPA) system, and are likely to be involved in the pathophysiology of anxiety disorders and major depression. We therefore investigated markers of serotonergic transmission and their modulation by chronic paroxetine in rats selectively bred for high (HAB) or low (LAB) anxiety-related behaviour, both under basal conditions and in response to emotional stress. Hippocampal serotonin 1 A (5-HT1A) receptor mRNA expression was reduced in HAB rats, whereas 5-HT concentrations in hippocampal microdialysates did not differ between HAB and LAB rats under basal conditions. In the hippocampus, overall expression of serotonin transporter binding sites was increased in HAB compared with LAB rats. Exposure to emotional stress failed to increase intrahippocampal 5-HT release in HAB rats whereas LAB rats displayed a physiological, albeit small rise. Chronic paroxetine treatment markedly increased the stress-induced rise in hippocampal 5-HT in HAB, but not LAB rats. This effect may be (at least in part) related to a greater down-regulation of hippocampal serotonin transporter binding sites by paroxetine in HABs compared with LABs, while 5-HT1A receptor expression remained unaffected in this brain area. The findings indicate reduced hippocampal serotonergic transmission in HAB rats as compared with LAB rats, which is evident both at the presynaptic (5-HT release) and the postsynaptic (5-HT1A receptor) level. Chronic paroxetine enhanced the presynaptic responsivity in HAB rats, but not LAB rats, pointing to a preferential efficacy of paroxetine in rats with enhanced anxiety/depression-related behaviour.     

Inositol phospholipid hydrolysis may mediate the action of proctolin on insect visceral muscle

The formation of inositol phosphates in response to the neuropeptide proctolin was studied in locust oviducts. Glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate were identified in the locust oviducts using anion-exchange chromatography. Proctolin stimulated the release of inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate during a 5-min incubation. In the presence of lithium ions the effects of proctolin were enhanced, with elevations of 98%, 42%, and 45% of inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate, respectively. Physiologically the effects of proctolin upon muscular contraction of locust oviducts were mimicked by the active phorbol ester, phorbol 12-myristate 13-acetate, and by the diacylglycerol analogue, 1-oleoyl-2-acetylglycerol. The inactive phorbol ester, 12-myristate 13-acetate 4-O-methyl ether, was without effect. The effects of the active phorbol ester and the diacylglycerol analogue were calcium-dependent requiring micromolar concentrations of calcium. The results indicate that the locust oviducts possess proctolin receptors that are linked to phosphatidylinositol metabolism and that inositol phospholipid hydrolysis may mediate the physiological action of proctolin.     

Deletion of the serotonin 5-HT2C receptor PDZ recognition motif prevents receptor phosphorylation and delays resensitization of receptor responses

Phosphorylation-deficient serotonin 5-HT(2C) receptors were generated to determine whether phosphorylation promotes desensitization of receptor responses. Phosphorylation of mutant 5-HT(2C) receptors that lack the carboxyl-terminal PDZ recognition motif (Ser(458)-Ser-Val-COOH; DeltaPDZ) was not detectable based on a band-shift phosphorylation assay and incorporation of (32)P. Treatment of cells stably expressing DeltaPDZ or wild-type 5-HT(2C) receptors with serotonin produced identical maximal responses and EC(50) values for eliciting [(3)H]inositol phosphate formation. In calcium imaging studies, treatment of cells expressing DeltaPDZ or wild-type 5-HT(2C) receptors with 100 nm serotonin elicited initial maximal responses and decay rates that were indistinguishable. However, a second application of serotonin 2.5 min after washout caused maximal responses that were approximately 5-fold lower with DeltaPDZ receptors relative to wild-type 5-HT(2C) receptors. After 10 min, responses of DeltaPDZ receptors recovered to wild-type 5-HT(2C) receptor levels. Receptors with single mutations at Ser(458) (S458A) or Ser(459) (S459A) decreased serotonin-mediated phosphorylation to 50% of wild-type receptor levels. Furthermore, subsequent calcium responses of S459A receptors were diminished relative to S458A and wild-type receptors. These results establish that desensitization occurs in the absence of 5-HT(2C) receptor phosphorylation and suggest that receptor phosphorylation at Ser(459) enhances resensitization of 5-HT(2C) receptor responses.     

Opposite effects of presynaptic 5-HT3 receptor activation on spontaneous and action potential-evoked GABA release at hippocampal synapses

5-HT(3) (serotonin type 3) receptors are targets of antiemetics, antipsychotics, and antidepressants and are believed to play a role in cognition. Nevertheless, contrasting results have been obtained with respect to their functions in the CNS and in the control of transmitter release. We used rat hippocampal neurons in single-neuron microcultures to identify the roles of presynaptic 5-HT(3) receptors at central synapses. 5-HT (10 microm) caused a transient > 10-fold increase in the frequency of miniature inhibitory postsynaptic currents without affecting amplitudes or kinetics. This effect was abolished by tropisetron (30 nm) and when Ca(2+) channels were blocked by 100 microm Cd(2+) it was mimicked and occluded when neurons were depolarized by 20 mm, but not 10 mm, K(+). Thus, activation of presynaptic 5-HT(3) receptors increased spontaneous GABA release by causing depolarization and opening of voltage-gated Ca(2+) channels. In microculture neurons, 5-HT transiently reduced action potential-evoked inhibitory autaptic currents by > 50%; this effect was blocked by tropisetron and mimicked by 20 mm, but not 10 mm, K(+). Miniature excitatory postsynaptic currents were not altered by 5-HT. Excitatory autaptic currents were tonically reduced, an effect attenuated by 5-HT(1A) antagonists. Thus, presynaptic 5-HT(3) receptors control GABA, but not glutamate, release and mediate opposite effects on spontaneous and action potential-dependent release.