Serotonin Transporter mRNA Levels Research Articles

T1727 Myosin Vi Is Necessary for Setting Brush Border NHE3 Activity: An Effect On NHE3 Trafficking/Endocytosis

Serotonin transporter (SERT) plays a critical role in regulating serotonin (5-HT) availability in the gut via reuptake of released serotonin. A decrease in SERT function and expression has been implicated in inflammatory bowel diseases and irritable colon. Hence, SERT is a potential pharmacological target in gastrointestinal disorders, however, very little is known regarding regulation of SERT in the human intestine. In this regard, EGF, a potent growth factor, is known to influence intestinal electrolyte and glucose transport processes and has protective effects on mucosa in models of colitis. Whether EGF regulates SERT function and expression in the human intestine is not known. The present studies were, therefore, designed to examine the regulation of SERT by EGF utilizing Caco2 cells grown on Transwell inserts as an In Vitro model. SERT activity was measured as Na+ and Cldependent 3[H] 5-HT uptake in fully differentiated cell monolayers. SERT mRNA abundance was determined by real time QRT-PCR. SERT promoter activity was assessed by luciferase assay in cells transiently transfected with promoter constructs. Treatment of Caco-2 cells with EGF from basolateral side (5-10ng/ml) for 24h significantly stimulated SERT activity (~50%, P<0.005). However, acute treatment with EGF (1-2 h) did not affect SERT function in Caco-2 cells. Corresponding with increased function at 24h, EGF treatment resulted in up-regulation of SERT mRNA levels (~1.8 fold) compared to control. For studying the regulation of SERT gene by EGF, a 873 bp (-871/+2) SERT promoter fragment was cloned upstream to the luciferase reporter gene and was found to be highly active when transfected in Caco2 cells. EGF treatment (10 ng/ml) for 8-24h resulted in significant stimulation in SERT promoter activity (~50-60%; P<0.05). Inhibition of EGF receptor (EGFR) tyrosine kinase activity by PD168393 (1nM) blocked the stimulatory effects of EGF on SERT promoter. Studies utilizing progressive deletion constructs of the SERT promoter indicated that the putative EGF responsive elements are in the -672/+472 region of the promoter. Conclusion: Our findings demonstrate transcriptional regulation of SERT by EGF via EGFR. These data define novel mechanisms of modulation of SERT function and expression in intestinal epithelium that may be relevant to therapeutic benefits of EGF in diarrheal diseases associated with altered 5-HT availability. [Supported by NIDDK/Dept of Veteran Affairs].

T1730 Curcumin Down-Regulates Niemann-Pick C1-Like 1 Expression in Intestinal Epithelial Cells

Serotonin transporter (SERT) plays a critical role in regulating serotonin (5-HT) availability in the gut via reuptake of released serotonin. A decrease in SERT function and expression has been implicated in inflammatory bowel diseases and irritable colon. Hence, SERT is a potential pharmacological target in gastrointestinal disorders, however, very little is known regarding regulation of SERT in the human intestine. In this regard, EGF, a potent growth factor, is known to influence intestinal electrolyte and glucose transport processes and has protective effects on mucosa in models of colitis. Whether EGF regulates SERT function and expression in the human intestine is not known. The present studies were, therefore, designed to examine the regulation of SERT by EGF utilizing Caco2 cells grown on Transwell inserts as an In Vitro model. SERT activity was measured as Na+ and Cldependent 3[H] 5-HT uptake in fully differentiated cell monolayers. SERT mRNA abundance was determined by real time QRT-PCR. SERT promoter activity was assessed by luciferase assay in cells transiently transfected with promoter constructs. Treatment of Caco-2 cells with EGF from basolateral side (5-10ng/ml) for 24h significantly stimulated SERT activity (~50%, P<0.005). However, acute treatment with EGF (1-2 h) did not affect SERT function in Caco-2 cells. Corresponding with increased function at 24h, EGF treatment resulted in up-regulation of SERT mRNA levels (~1.8 fold) compared to control. For studying the regulation of SERT gene by EGF, a 873 bp (-871/+2) SERT promoter fragment was cloned upstream to the luciferase reporter gene and was found to be highly active when transfected in Caco2 cells. EGF treatment (10 ng/ml) for 8-24h resulted in significant stimulation in SERT promoter activity (~50-60%; P<0.05). Inhibition of EGF receptor (EGFR) tyrosine kinase activity by PD168393 (1nM) blocked the stimulatory effects of EGF on SERT promoter. Studies utilizing progressive deletion constructs of the SERT promoter indicated that the putative EGF responsive elements are in the -672/+472 region of the promoter. Conclusion: Our findings demonstrate transcriptional regulation of SERT by EGF via EGFR. These data define novel mechanisms of modulation of SERT function and expression in intestinal epithelium that may be relevant to therapeutic benefits of EGF in diarrheal diseases associated with altered 5-HT availability. [Supported by NIDDK/Dept of Veteran Affairs].

T1726 Epidermal Growth Factor (EGF) Stimulates Serotonin Transporter Function and Expression via Transcriptional Mechanisms in Human Intestinal Epithelial Cells

Serotonin transporter (SERT) plays a critical role in regulating serotonin (5-HT) availability in the gut via reuptake of released serotonin. A decrease in SERT function and expression has been implicated in inflammatory bowel diseases and irritable colon. Hence, SERT is a potential pharmacological target in gastrointestinal disorders, however, very little is known regarding regulation of SERT in the human intestine. In this regard, EGF, a potent growth factor, is known to influence intestinal electrolyte and glucose transport processes and has protective effects on mucosa in models of colitis. Whether EGF regulates SERT function and expression in the human intestine is not known. The present studies were, therefore, designed to examine the regulation of SERT by EGF utilizing Caco2 cells grown on Transwell inserts as an In Vitro model. SERT activity was measured as Na+ and Cldependent 3[H] 5-HT uptake in fully differentiated cell monolayers. SERT mRNA abundance was determined by real time QRT-PCR. SERT promoter activity was assessed by luciferase assay in cells transiently transfected with promoter constructs. Treatment of Caco-2 cells with EGF from basolateral side (5-10ng/ml) for 24h significantly stimulated SERT activity (~50%, P<0.005). However, acute treatment with EGF (1-2 h) did not affect SERT function in Caco-2 cells. Corresponding with increased function at 24h, EGF treatment resulted in up-regulation of SERT mRNA levels (~1.8 fold) compared to control. For studying the regulation of SERT gene by EGF, a 873 bp (-871/+2) SERT promoter fragment was cloned upstream to the luciferase reporter gene and was found to be highly active when transfected in Caco2 cells. EGF treatment (10 ng/ml) for 8-24h resulted in significant stimulation in SERT promoter activity (~50-60%; P<0.05). Inhibition of EGF receptor (EGFR) tyrosine kinase activity by PD168393 (1nM) blocked the stimulatory effects of EGF on SERT promoter. Studies utilizing progressive deletion constructs of the SERT promoter indicated that the putative EGF responsive elements are in the -672/+472 region of the promoter. Conclusion: Our findings demonstrate transcriptional regulation of SERT by EGF via EGFR. These data define novel mechanisms of modulation of SERT function and expression in intestinal epithelium that may be relevant to therapeutic benefits of EGF in diarrheal diseases associated with altered 5-HT availability. [Supported by NIDDK/Dept of Veteran Affairs].

Serotonin transporter mRNA levels are associated with the methylation of an upstream CpG island

The serotonin reuptake transporter (5HTT) is thought to be the principal regulator of serotonergic activity and epigenetic effects at this locus are thought to be important moderators of vulnerability to neuropsychiatric illness. In attempt to understand the basis of this regulation, several gene polymorphisms that affect 5HTT mRNA levels have been described. But to date, no clear mechanism linking these polymorphisms to vulnerability to epigenetic effects have been described. In this communication, we describe a CpG island in the 5' region of the 5HTT gene that contains an alternative exon 1 and possible promoter for 5HTT. We then confirm the existence of this transcript and ascertain the methylation status of this CpG island in 49 lymphoblast cell lines and analyze the relationship between methylation and 5HTT mRNA levels. We demonstrate that methylation at this CpG island is associated with decreased levels of 5HTT mRNA, but that this effect is evident only when 5HTTLPR genotype is taken into account. We suggest that these findings have significant implications for the understanding of the role of this locus in behavioral illness.

Chronic intraperitoneal injection of interferon‐α reduces serotonin levels in various regions of rat brain, but does not change levels of serotonin transporter mRNA, nitrite or nitrate

Interferon-alpha therapy is associated with a high rate of depression, but the pathophysiological mechanisms remain unclear. The purpose of the present study was to investigate the effects of i.p. administered interferon-alpha on monoaminergic neurotransmission in the brain. The levels of monoamines and associated metabolites were measured in various regions of the rat brain using a high-performance liquid chromatography-electrochemical detection system. The serotonin transporter mRNA levels were also measured using in situ hybridization. After 1 day, dopamine turnover was diminished in the cortex. Norepinephrine turnover was decreased in most regions tested after 4 days. However, these changes were transient. After 14 days, serotonin turnover was increased in the frontal cortex and hippocampus in rats given a dose of 20 000 IU/kg; in the frontal cortex, hippocampus, amygdala, thalamus, hypothalamus and brainstem in those on 200 000 IU/kg; and in the thalamus and hypothalamus in those on 2 000 000 IU/kg (all P < 0.05). However, 14-day treatment did not significantly change serotonin transporter mRNA levels. Next, the question of whether interferon-alpha affects monoamine levels via induction of nitric oxide (NO), was investigated. However, there were no changes in either NO2- or NO3-, as markers of NO production, in any brain regions after 14-day treatment. These results suggest that chronic peripheral administration of interferon-alpha induces metabolic changes in the central serotonin system. Further investigation is needed to determine exactly how this cytokine affects the central serotonin system and to assess whether a central serotonin abnormality is involved in interferon-induced depression.

Allelic expression of serotonin transporter (SERT) mRNA in human pons: lack of correlation with the polymorphism SERTLPR

An insertion/deletion polymorphism in the SERT linked promoter region (SERTLPR), previously reported to regulate mRNA expression in vitro, has been associated with mental disorders and response to psychotropic drugs. Contradictory evidence, however, has raised questions about the role of SERTLPR in regulating mRNA expression in vivo. We have used analysis of allelic expression imbalance (AEI) of SERT mRNA to assess quantitatively the contribution of SERTLPR to mRNA expression in human post-mortem pons tissue sections containing serotonergic neurons of the dorsal and median raphe nuclei. Any difference in the expression of one allele over the other indicates the presence of cis-acting elements that differentially affect transcription and/or mRNA processing and turnover. Using a marker SNP in the 3' untranslated region of SERT mRNA, statistically significant differences in allelic mRNA levels were detected in nine out of 29 samples heterozygous for the marker SNP. While the allelic expression differences were relatively small (15-25%), they could nevertheless be physiologically relevant. Although previous results had suggested that the long form of SERTLPR yields higher mRNA levels than the short form, we did not observe a correlation between SERTLPR and allelic expression ratios. Also in contrast to previous results, we found no correlation between SERTLPR and allelic expression ratios or SERT mRNA levels in B-lymphocytes. This study demonstrates that regulation of SERT mRNA is independent of SERTLPR, but could be associated with polymorphisms in partial linkage disequilibrium with SERTLPR.

Allelic Isoforms and Decrease in Serotonin Transporter mRNA in Lymphocytes of Patients with Major Depression

Serotonin transporter, measured by the specific binding of [³H]paroxetine, has been reported to be reduced in circulating lymphocytes of patients with major depression. Due to this observation, the objective of the present report was to determine the levels of serotonin transporter mRNA in lymphocytes obtained from 29 major depression patients (4 men, age 33.10 ± 1.63 years) and from 30 subjects included as a control group (4 men, age 37.54 ± 2.18 years) using RT-PCR. The patients were diagnosed according to the criteria of the American Psychiatric Association, and had a severity of depression of 32.68 ± 1.55 determined by the Hamilton Rating Scale for Depression. The DNA was submitted to polymerase chain reaction with primers for the 5′ regulatory region of human serotonin transporter, which could show the long and the short allelic forms of the transporter gene for the 5HTTLPR polymorphism. Semiquantitative analysis was performed using β-actin as internal and external standard. Control subjects presented the two allelic forms in 9.09% and depressed patients in 8.69%. The long variant was present in 73% of controls and in 60% of patients, without significant differences. There was a significant reduction in mRNA in depressed patients expressing the long allele. The number of immunofluorescent lymphocytes, labeled with a specific antibody against serotonin transporter, was reduced in the patients, as well as CD3+ lymphocytes. Serotonin and 5-hydroxyindoleacetic acid in platelet-poor plasma or lymphocytes did not differ between depressed patients and controls. The reduction in lymphocyte serotonin transporter described in major depression might be due to a decrease in the level of its mRNA and in the number of cells expressing it. These observations might implicate that functional modifications are associated with nervous-immune interactions in depression.

Rat brain serotonin neurones that express neuronal nitric oxide synthase have increased sensitivity to the substituted amphetamine serotonin toxins 3,4-methylenedioxymethamphetamine and p-chloroamphetamine

Substituted amphetamines such as p-chloroamphetamine and the abused drug methylenedioxymethamphetamine cause selective destruction of serotonin axons in rats, by unknown mechanisms. Since some serotonin neurones also express neuronal nitric oxide synthase, which has been implicated in neurotoxicity, the present study was undertaken to determine whether nitric oxide synthase expressing serotonin neurones are selectively vulnerable to methylenedioxymethamphetamine or p-chloroamphetamine. Using double-labeling immunocytochemistry and double in situ hybridization for nitric oxide synthase and the serotonin transporter, it was confirmed that about two thirds of serotonergic cell bodies in the dorsal raphé nucleus expressed nitric oxide synthase, however few if any serotonin transporter immunoreactive axons in striatum expressed nitric oxide synthase at detectable levels. Methylenedioxymethamphetamine (30mg/kg) or p-chloroamphetamine (2×10mg/kg) was administered to Sprague–Dawley rats, and 7 days after drug administration there were modest decreases in the levels of serotonin transporter protein in frontal cortex, and striatum using Western blotting, even though axonal loss could be clearly seen by immunostaining. p-Chloroamphetamine or methylenedioxymethamphetamine administration did not alter the level of nitric oxide synthase in striatum or frontal cortex, determined by Western blotting. Analysis of serotonin neuronal cell bodies 7 days after p-chloroamphetamine treatment, revealed a net down-regulation of serotonin transporter mRNA levels, and a profound change in expression of nitric oxide synthase, with 33% of serotonin transporter mRNA positive cells containing nitric oxide synthase mRNA, compared with 65% in control animals. Altogether these results support the hypothesis that serotonin neurones which express nitric oxide synthase are most vulnerable to substituted amphetamine toxicity, supporting the concept that the selective vulnerability of serotonin neurones has a molecular basis.

Increase in expression of brain serotonin transporter and monoamine oxidase a genes induced by repeated experience of social defeats in male mice.

Serotonin transporter and monoamine oxidase (MAO) A are involved in the inactivation of serotonin. The former is responsible for serotonin re-uptake from the synapse, whereas the latter catalyzes serotonin deamination in presynaptic terminals. Expression of serotonin transporter and MAO A genes was investigated in raphe nuclei of midbrain of CBA/Lac male mice with repeated experience of social victories or defeats in 10 daily aggressive confrontations. The amount of cDNA of these genes was evaluated using multiplex RT-PCR. Two independent experiments revealed that the defeated mice were characterized by significantly higher levels of serotonin transporter and MAO A mRNAs than the control and aggressive animals. Increased expression of MAO A and serotonin transporter genes is suggested to reflect the accelerated serotonin degradation in response to activation of the serotonergic system functioning induced by social stress. Significant positive correlation between MAO A and serotonin transporter mRNA levels suggests common pathways of regulation of transcriptional activity of these genes.

Repeated experience of social defeats increases serotonin transporter and monoamine oxidase A mRNA levels in raphe nuclei of male mice

Serotonin transporter (SERT) and monoamine oxidase A (MAOA) mRNA levels in the raphe nuclei area of the midbrain were measured by the multiplex reverse transcription-polymerase chain reaction method in male mice with repeated experience of social victories (winners) and defeats (losers) in ten daily agonistic confrontations. Experiments revealed enhanced SERT and MAOA mRNA levels in the losers compared with the winners and controls. It has been supposed that SERT and MAOA genes are involved in enhancement of serotonin inactivation in response to the increase of serotonergic activity shown earlier in the losers. A positive correlation between MAOA and SERT mRNA levels in the raphe nuclei area of the midbrain was shown.

Gender differences in acute and chronic stress in Wistar Kyoto (WKY) rats.

While females are considered more susceptible to depressive behavior, this assertion is not strongly supported by the experimental literature. Since stress contributes to depressive behavior, male and female Wistar Kyoto (WKY) rats were exposed to either one session (acute stress) or 5 sessions (chronic stress) of restraint plus cold in order to study depressive behavior in male and female rats. After their respective treatment exposure, rats were tested in the open field test (OFT) and for retention of a passive-avoidance (P-A) task. One stress session resulted in significant immobility in the OFT for males, whereas 5 sessions were required to produce similar immobility in female rats. Acute stress interfered with the retention of the P-A response for males, while both acute and chronic stress produced poor P-A responses in female rats. Food consumption decreased progressively, as a function of stress sessions, in female rats, whereas feeding in males returned to control levels after five stress days. Both acute and chronic stress exacerbated the stress ulcer response in male rats, but not in female rats. Chronic, but not acute, stress resulted in an increase in serotonin transporter mRNA levels in the dorsal raphe nucleus of both male and female rats. The general consensus from these data suggested that female rats were more vulnerable to chronic stress and consequently supported the notion that females may be more susceptible to stress-induced behavioral depression. Key Words: WKY rats, acute and chronic stress, gender, passive avoidance, open field behavior, stress-ulcer, adrenal weight, serotonin, dorsal raphe nucleus

Reserpine modulates serotonin transporter mRNA levels in the rat brain

Despite recent advances in determining central serotonin (5-HT) function, the basic aspects by which serotonin neurotransmission is controlled and regulated are still not understood. Since the serotonin transporter (5-HTT) is involved in terminating the action of 5-HT that is released from the presynaptic nerve terminal, the regulation of 5-HTT may be an important step in controlling 5-HT neurotransmission at the synaptic cleft. The present study investigated the effects of reserpine administration on 5-HTT gene expression as well as on tryptophan hydroxylase (TPH) expression in the rat brain. Male Sprague-Dawley rats were injected with reserpine (10 mg kg , i.p.) and sacrificed at 8 h, 3 days, 7 days or 21 days after the injection. Control rats were injected with saline and sacrificed either at 8 h or 21 days after the injection. The midbrain region was dissected, RNA was isolated and probed for 5-HTT expression using Northern Blotting. Data were analyzed using Super-Anova followed by posthoc Dunnett's test. While mRNA levels for 5-HTT were unchanged at 8 h after reserpine, a significant decrease was noted at 3 days and 7 days (F = 10; p < 0.0001). 5-HTT mRNA levels returned to control levels by 21 days. In contrast, TPH expression was unaltered at all time points examined. The results of this study provide useful information regarding the role that the 5-HTT may be playing in the homeostatic control of 5-HT neurotransmission at the synapse.

Effects of interferon- α, interferon- γ and cAMP on the transcriptional regulation of the serotonin transporter

We examined the effects of interferon- α and - γ, which are known to have psychiatric side effects including depression, on the transcriptional regulation of the serotonin transporter and the uptake activity of the serotonin transporter in order to clarify the involvement of the serotonin transporter in the pathogenesis of interferon-induced depression. In human placental choriocarcinoma cells (BeWo cells), both messenger RNA (mRNA) for the serotonin transporter and the imipramine-sensitive uptake of serotonin were detected. The levels of serotonin transporter mRNA were increased by treatment with interferon- α and - γ for 3 h. The increase in serotonin transporter mRNA elicited by the interferons was inhibited by treatment with actinomycin D, an inhibitor of transcription. Treatment with interferon- α or - γ for 3–6 h, but not for 30 min, increased the uptake activity of the serotonin transporter. Treatment with dibutyryl cAMP (Dib-cAMP) which was reported to up-regulate the transcription of the serotonin transporter, also increased the mRNA levels and the activity of serotonin transporter in BeWo cells. The levels of serotonin transporter mRNA gradually increased after treatment with Dib-cAMP over 24 h, while the maximal increase in serotonin transporter mRNA elicited by the interferons was detected 3 h after the treatment. The level of serotonin transporter mRNA was increased both in the midbrain and adrenal glands of mice which were treated with interferons for 3 h. These results suggest that the interferon-induced psychiatric side effects arise through regulation of serotonin transporter transcription and that the transcriptional regulation of the serotonin transporter is a possible neurochemical mechanism of affective disorders.

Cocaine, ethanol, and genotype effects on human midbrain serotonin transporter binding sites and mRNA levels.

Earlier platelet and postmortem brain studies have found alterations in serotonin transporter function in ethanol-abusing human subjects. The present investigation tested the hypothesis that brain serotonin transporter function is altered in chronic users of ethanol and cocaine, which might be related to a common serotonin transporter promoter polymorphism. Serotonin transporter binding sites, serotonin transporter mRNA levels, and serotonin transporter promoter variants were quantified in postmortem samples from a group of human subjects who had been ethanol users or cocaine users and then compared to those of a matched group of comparison subjects. Quantitative autoradiographic and in situ hybridization assays were performed in midbrain samples that contained the dorsal and median raphe nuclei (the location of serotonin cell bodies that innervate the forebrain). There was a significant overall cocaine-by-ethanol-by-genotype interaction. Dorsal raphe [125I]CIT binding to the serotonin transporter was lower in cocaine users than in comparison subjects. In addition, serotonin transporter binding and serotonin transporter mRNA levels varied significantly by genotype. It was also found that serotonin transporter binding in subjects with either the short or heterozygote genotype was significantly higher in the ethanol-user subjects. Serotonin transporter binding sites were regulated in a region-specific and substance-specific pattern, which was not simply a local response to functional blockade. Also, a reciprocal relationship appeared to exist between cocaine and ethanol effects in the dorsal raphe, which may have interesting clinical implications for dual-diagnosis patients. It is possible that serotonin transporter promoter genotype may play a complex role in chronic ethanol dependence.

Estradiol-17 beta increases serotonin transporter (SERT) mRNA levels and the density of SERT-binding sites in female rat brain.

The aim of the present study was to determine the effect of estradiol-17 beta (E2), in its positive feedback mode for gonadotropin release, on the serotonin transporter (SERT) in female rat brain. Levels of SERT mRNA were determined by in situ hybridization and SERT-binding sites were measured by quantitative [3H]paroxetine receptor autoradiography. The injection of estradiol benzoate (EB) in acutely ovariectomized rats increased significantly (approximately 50%) the numbers of cells that expressed SERT mRNA in the dorsal raphe nucleus and the density of SERT-binding sites in lateral septum (90%), basolateral amygdala (20%), ventral nucleus of thalamus (250%) and ventromedial hypothalamic nucleus (250%). SERT-binding sites in EB-treated rats were significantly lower in periaqueductal central grey (15%). These findings indicate that effects on SERT gene expression may be involved in the E2-induction of the gonadotropin surge. Together with our previous findings, they also suggest that the sex differences in depression and the apparent psychotropic effect of E2 may be due to the action of E2 on the serotonin transporter as well as 5-HT2A receptors.

Abnormal expression of serotonin transporter mRNA in the frontal and temporal cortex of schizophrenics.

Postmortem brain tissue was used to measure expression of serotonin transporter mRNA in the left frontal and left temporal cortex from schizophrenics (n = 24) and from normal control individuals with no history of psychiatric illness (n = 10). There was an approximately four-fold increase of serotonin transporter mRNA in Brodmann's area 9 and a two-fold decrease in Brodmann's areas 21 and 22. The changes in serotonin transporter mRNA were more prominent in schizophrenics who had received neuroleptic drugs within 1 week prior to death than in schizophrenics who were neuroleptic-free for more than 2 weeks prior to death, suggesting that the changes may be related to neuroleptic treatment. There was no correlation between serotonin transporter mRNA levels and the sex or age of schizophrenic and control cases or the postmortem delay intervals. These results support the hypothesis that region-specific changes in serotonin transporter expression occur in brains of some schizophrenics. Future studies are necessary to determine whether the changes in serotonin transporter expression are drug-related or are associated with the illness itself.

P‐Chlorophenylalanine Changes Serotonin Transporter mRNA Levels and Expression of the Gene Product

After a single intraperitoneal injection of the irreversible tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA; 300 mg/kg), there was a rapid down-regulation of serotonin (5-HT) transporter mRNA levels in cell bodies. This change was significant at 1 and 2 days after PCPA administration within the ventromedial but not the dorsomedial portion of the dorsal raphe nucleus. Seven days after PCPA treatment, 5-HT transporter mRNA levels were significantly elevated compared with controls in both regions of the dorsal raphe nucleus. PCPA administration produced no change in the [3H]citalopram binding and synaptosomal [3H]5-HT uptake in terminal regions at 2 and 7 days after treatment but significantly reduced both these parameters by approximately 20% in the hippocampus and in cerebral cortex 14 days after PCPA administration. The striatum showed a lower sensitivity to this effect. No significant changes were observed in the levels of [3H]citalopram binding to 5-HT cell bodies in the dorsal raphe nucleus. In the same animals used for 5-HT transporter mRNA level measurements, levels of tryptophan hydroxylase mRNA in neurons of the ventromedial and dorsomedial portions of the dorsal raphe nucleus were increased 2 days after PCPA administration and fell to control levels 7 days after injection in the ventromedial region but not in the dorsomedial portion of the dorsal raphe nucleus, where they remained significantly higher than controls. Altogether, these results show that changes in 5-HT transporter mRNA are not temporally related to changes in 5-HT transporter protein levels. In addition, our results suggest that the 5-HT transporter and tryptophan hydroxylase genes are regulated by different mechanisms. We also provide further evidence that dorsal raphe 5-HT neurons are differentially regulated by drugs, depending on their location.

Quantitative RT-PCR distribution of serotonin 5-HT6 receptor mRNA in the central nervous system of control or 5,7-dihydroxytryptamine-treated rats.

Possible adaptive changes of the recently cloned serotonin 5-HT6 receptor after the selective lesion of serotoninergic neurons by an intracerebral administration of 5,7-dihydroxytryptamine were investigated using competitive RT-PCR (reverse transcription followed by polymerase chain reaction) for the measurement of 5-HT6-mRNA in various areas of the rat central nervous system. In control rats, 5-HT6-mRNA was the most abundant in the nucleus accumbens, followed by the olfactory tubercle and the striatum. High levels of 5-HT6-mRNA were also found in the hypothalamus and the hippocampus, whereas the cerebral cortex, the substantia nigra, and the spinal cord contained moderate levels of the transcript. Low but easily quantifiable levels of 5-HT6-mRNA were measured in the ventral tegmental area, the anterior raphe area, and the cerebellum. In addition, moderate to low levels of this mRNA were also found in dorsal root ganglia and the pituitary gland. Three weeks after the microinfusion of 5,7-dihydroxytryptamine into the anteroventral vicinity of the dorsal raphe nucleus in nomifensine-pretreated rats, the levels of serotonin transporter-mRNA were reduced by 90% in the anterior raphe area, as expected of the extensive lesion of serotoninergic neurons. In contrast, quantitative determinations of the 5-HT6-mRNA in this area as well as in the nucleus accumbens, the striatum, and the hippocampus indicated that its levels were not significantly different in 5,7-dihydroxytryptamine-treated rats and in controls. These data showed that the 5-HT6 receptor: 1) is not an autoreceptor, and 2) exhibits probably no up regulation in postsynaptic target cells after the selective degeneration of serotoninergic projections.

Serotonin depletion decreases serotonin transporter mRNA levels in rat brain

In order to study the impact of serotonin depletion on gene expression of the serotonin transporter (5-HT t) we measured 5-HT t mRNA levels by Northern blot in rats treated with p-chlorophenylalanine methyl ester (PCPA) for 10 days. Six rats received PCPA i.p. only, and another 6 rats receiving 0.9% NaCl served as controls. An additional group of 6 rats received both PCPA i.p. and imipramine, 5 mg/kg/day by osmotic minipumps. 5-HT t mRNA levels decreased to 81.1% ( P = 0.05) and 76.0% ( P = 0.05) of the control level for PCPA treated animals without and with concomitant imipramine treatment, respectively. The average level of the PCPA treated groups was 78.6% ( P = 0.03). The isolated effect of 21 days of imipramine treatment was a 5-HT t mRNA level of 89.4%, which was not significantly different from the control level. In conclusion, 5_HT t gene expression is suppressed in the serotonin depleted state. A decreased synaptic reuptake of 5-HT may be interpreted as a compensatory mechanism aiming at preserving adequate synaptic 5-HT levels in a generally deficient state.

Regulation of the human serotonin transporter. Cholera toxin-induced stimulation of serotonin uptake in human placental choriocarcinoma cells is accompanied by increased serotonin transporter mRNA levels and serotonin transporter-specific ligand binding.

Treatment of confluent cultures of JAR human placental choriocarcinoma cells with cholera toxin or forskolin for 16 h markedly stimulated (2.4-fold) serotonin transport activity in these cells. Cycloheximide, an inhibitor of protein synthesis or actinomycin D, an inhibitor of mRNA synthesis effectively blocked this stimulation. Northern blot analysis revealed that treatment with cholera toxin resulted in severalfold increase in the concentrations of the three mRNA species (6.8, 4.9 and 3.0 kilobases in size) which hybridized to the human placental serotonin transporter cDNA. Under similar conditions, the concentrations of the mRNA species which hybridized to the human placental taurine transporter cDNA or to the human beta-actin cDNA were not affected. Analysis of paroxetine-sensitive binding of the cocaine analog 2 beta-carbomethoxy-3 beta-(4- [125I]iodophenyl)tropane to the membranes prepared from control and cholera toxin-treated cells indicated that the maximal binding capacity was increased 2.5-fold by cholera toxin, with no significant change in the binding affinity. Thus, stimulation of serotonin transporter activity in the placental choriocarcinoma cells following cholera toxin treatment is likely a result of an increase in cell surface density of the serotonin transporter protein as a consequence of increased steady state serotonin transporter mRNA levels.