Serotonin Transporter mRNA Research Articles

Pharmacological characterization and visualization of the glial serotonin transporter

Astrocytes contain transport systems that are capable of removing various neurotransmitters from the synaptic cleft by transporters present in the plasma membrane. Glial serotonin transporter (SERT) plays an important role in the re-uptake of 5-hydroxytryptamine (5-HT). We examined the pharmacological characterization of 5-HT uptake into rat cortical synaptosomes and cultured rat astrocytes, and the immunodetection of glial SERT proteins using specific site-directed monoclonal antibodies (MoAb). Furthermore, using a reverse transcriptase-polymerase chain reaction (RT-PCR) method, we addressed the expression of SERT mRNA in cultured rat astrocytes. We investigated the inhibitory effects of various monoamine uptake inhibitors on the uptake of [ 3H]5-HT into cultured astrocytes and cortical synaptosomes. Tricyclic antidepressants (clomipramine and imipramine) as well as selective serotonin re-uptake inhibitors (fluvoxamine, fluoxetine and zimelidine) were very potent inhibitors of [ 3H]5-HT uptake in both preparations. In contrast, the inhibitory effects of NE uptake inhibitors (nisoxetine and desipramine) and cocaine were weaker than those of 5-HT uptake inhibitors. In addition, dopamine (DA) uptake inhibitors (nomifensine and GBR-12935) exhibited a Ki value in the low micromolar range. The inhibitory potencies were in the order 5-HT uptake inhibitors (clomipramine, fluvoxamine, fluoxetine, imipramine and zimelidine)>NE uptake inhibitors (nisoxetine and desipramine)=cocaine>DA uptake inhibitors (nomifensine and GBR-12935). There was no difference in the order of the inhibitory effects of various monoamine uptake inhibitors between the two preparations. A correlation analysis of the potencies of various monoamine uptake inhibitors in the inhibition of [ 3H]5-HT into cultured astrocytes and cortical synaptosomes produced a highly significant correlation coefficient of 0.9893 ( P<0.0001). Immunocytochemical staining using anti-SERT MoAb in cultured astrocytes revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or golgi membranes, showed a considerable level of immunoreactivity. Extracts of astrocytes and synaptosomes from the cortex were immunoblotted with anti-SERT MoAb. SDS-PAGE/Western blots indicate that anti-SERT MoAb recognized two bands of 120 and 73 kDa in both preparations. RT-PCR demonstrated that astrocytes in cultured expressed mRNA for the cloned SERT protein, which has been characterized as the neuronal SERT. These pharmacological experiments indicate that this uptake process takes place through glial SERT that is very similar to neuronal SERT. Furthermore, the present data also indicate that the presence of the mRNA and protein for the neuronal SERT were established in cultured rat astrocytes, and the polypeptide portion of SERT in astrocytes and frontal cortex could be the same gene product.

Regulation of serotonin transporter gene expression in human glial cells by growth factors

The aims of this study were to identify monoamine transporters expressed in human glial cells, and to examine the regulation of their expression by stress-related growth factors. The expression of serotonin transporter mRNA was detected by reverse transcriptase-polymerase chain reaction in normal human astrocytes, whereas the dopamine transporter (DAT) and the norepinephrine transporter (NET) were not detected. The cDNA sequence of the “glial” serotonin transporter in astrocytes was consistent with that reported for the “neuronal” serotonin transporter (SERT). Moreover, we also demonstrated SERT expression in glial fibrillary acidic protein-positive cells by immunocytochemical staining in normal human astrocytes. Serotonin transporter gene expression was also detected in glioma-derived cell lines (A172, KG-1-C and KGK). Addition of basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) for 2 days increased serotonin transporter gene expression in astrocytes and JAR (human choriocarcinoma cell line). Basic fibroblast growth factor, but not epidermal growth factor, increased specific [ 3H]serotonin uptake in astrocytes in a time (1–4 days)- and concentration (20–100 ng/ml)-dependent manner. The expression of genes for basic fibroblast growth factor and epidermal growth factor receptors was detected in astrocytes. These findings suggest that the expression of the serotonin transporter in human glial cells is positively regulated by basic fibroblast growth factor.

Electroconvulsive shock regulates serotonin transporter mRNA expression in rat raphe nucleus.

The antidepressive actions of electroconvulsive shock (ECS) therapy are considered to involve altered neurotransmission of serotonin. In this study, we investigated the effects of acute and chronic ECS on 5-hydroxytryptamine (5-HT) transporter mRNA expression in rat raphe nucleus. We found that serotonin transporter (5-HTT) mRNA expression was decreased in 9 and 24 h after acute ECS and in 3, 9, 24 h and 2 weeks after chronic ECS in rat raphe nucleus. We presume that the adaptive change in 5-HTT mRNA expression is possibly related to the therapeutic efficacy of electroconvulsive therapy (ECT) on medication-resistant depression.

Serotonergic Neurons Are Targets for Leptin in the Monkey1

Leptin is a secretory product of adipocytes that has been shown to affect food intake, metabolism, and reproduction. One site of leptin's action is the central nervous system, where the leptin receptor (Ob-R) messenger ribonucleic acid (mRNA) and protein are expressed in discrete areas. In both the rat and monkey, Ob-R mRNA has been localized in the Raphe nuclei of the brainstem. Neurons in the Raphe nuclei are the primary source of serotonin in the brain. Serotonergic pathways influence both feeding and reproduction, and these cells are plausible direct targets for leptin's action. We used double label in situ hybridization and computerized image analysis to determine whether serotonergic neurons in the brainstem of the female pigtailed macaque (Macaca nemestrina) express Ob-R mRNA. We observed that many cells in the Raphe nuclei express serotonin transporter mRNA, a marker of serotonergic cells, and Ob-R mRNA. Based on quantitative analysis, the highest number of cells that express both serotonin transporter and Ob-R mRNAs were found in the caudal dorsal Raphe and median Raphe nuclei; fewer double labeled cells were situated in the caudal linear nucleus and rostral median Raphe, whereas double labeled cells occurred infrequently in the rostral dorsal Raphe. These observations suggest that leptin may act on serotonergic cells to mediate some of its effects on ingestive behavior, metabolism, and reproduction.

Opposing changes in serotonin and norepinephrine transporter mRNA levels after serotonin depletion

We and others have earlier shown that severe serotonin depletion leads to a compensatory down-regulation in the expression of the serotonin transporter (5HTT) gene. We have now investigated the expression of both the 5HTT and the norepinephrine transporter (NET) gene to assess the possible interaction between the noradrenergic and the serotonergic neurotransmitter systems. Acute severe serotonin depletion induced by p-chlorophenylalanine (PCPA) treatment leads to enhanced NET Long mRNA levels and reduced 5HTT mRNA level. This change in transporter mRNA expression was paralleled by a non-significant change in protein expression. Chronic severe serotonin depletion combined with treatment with the antidepressant imipramine leads to enhanced NET Long mRNA levels. Acute treatment with the monoamine oxidase A inhibitor clorgyline, acute moderate NE reduction (α-methyl- p-tyrosine treatment) or less severe depletion for 3 weeks have no effect on the gene expression of the transporters. Taken together, the present data demonstrate that the NET gene expression is enhanced in case of severe serotonin depletion.

Cocaine self-administration in rats differentially alters mRNA levels of the monoamine transporters and striatal neuropeptides

The potential neuroadaptations to cocaine self-administration (SA) were evaluated using quantitative in situ hybridisation histochemistry. Levels of mRNAs of the monoamine transporters, i.e. the primary molecular targets of cocaine, and the striatal neuropeptides substance P and enkephalin, which predominantly exist in different populations of dopaminoceptive striatal neurons, were quantified in rats which had reached different stages of acquisition of cocaine SA. Thus, animals were killed 1 h after completing a self-administration session (i) early in or after acquisition of cocaine SA (ii) after various regimes of chronic cocaine SA, and (iii) a 10-day period of withdrawal from chronic cocaine intake. Control mRNA levels of all molecules under study were those quantified in animals receiving i.v. saline yoked to rats self-administering cocaine (1.5 or 0.75 mg/kg per infusion, depending on the experiment). Monoamine transporter expression was differentially altered by cocaine; dopamine transporter mRNA levels in the ventral tegmental area, but not in the substantia nigra, were increased following withdrawal from cocaine, suggesting a role for the upregulated mesolimbic dopamine transporter in the mechanisms underlying relapse to cocaine taking. By contrast, serotonin transporter mRNA in the dorsal raphé and noradrenaline transporter mRNA in the locus coeruleus remained unaltered under all experimental conditions. In addition, the expression of the striatal neuropeptides was also differentially altered; substance P mRNA levels were transiently increased in the shell of the nucleus accumbens by prolonged cocaine self-administration, but enkephalin mRNA levels in the dorsal and ventral striatum remained unaltered under all conditions.

Acute stress decreases serotonin transporter mRNA in the raphe pontis but not in other raphe nuclei of the rat

In addition to elevated corticosterone levels, stress produces structural changes and neuronal damage especially in the hippocampus. In this line it has been shown, that in rats single or repeated immobilisation markedly reduces brain-derived neurotrophic factor (BDNF) mRNA levels in the hippocampal formation. Since this neurotrophin also controls the efficacy of serotonergic neurotransmission, the aim of the current study was to investigate the effect of acute immobilization stress on the expression of serotonin transporter (SERT) mRNA in the raphe nuclei as a parameter of serotonergic innervation. We have examined the expression of SERT mRNA and of BDNF mRNA in rats upon acute immobilisation by quantitative in situ hybridisation with a 35S-labelled oligonucleotide probe. Elevated corticosterone levels in stressed animals confirmed as internal controls the effect of stress under our conditions. Acute stress led to a significant decrease of BDNF mRNA in the hippocampus and of SERT mRNA in the raphe pontis, but not in other raphe nuclei investigated. These data provide evidence for fast interactions between neurotrophins, corticosterone and serotonergic neurotransmission under stress conditions.

Localization of calcitonin receptor mRNA in the mouse brain: coexistence with serotonin transporter mRNA

To elucidate the sites of and mechanisms of analgesic effect of centrally injected calcitonin, we examined expression of calcitonin receptor mRNA in the mouse brain by in situ hybridization techniques. Calcitonin receptor mRNA was expressed in various brain regions, including the preoptic area, dorsomedial hypothalamic nucleus, lateral hypothalamic area, periaqueductal gray, dorsal raphe nucleus, locus coeruleus, lateral parabrachial nucleus, gigantocellular reticular nucleus alpha part, lateral paragigantocellular nucleus, raphe magnus nucleus and solitary tract nucleus, which are known to play important roles in pain modulation. In addition, a double in situ hybridization technique demonstrated the intense expression of calcitonin receptor mRNA on serotonergic neurons in some raphe nuclei and the lateral paragigantocellular nucleus, suggesting the involvement of central serotonergic pathways in analgesic effect of calcitonin.

Effects of Chronic Antidepressant Treatments on Serotonin Transporter Function, Density, and mRNA Level

To investigate functional changes in the brain serotonin transporter (SERT) after chronic antidepressant treatment, several techniques were used to assess SERT activity, density, or its mRNA content. Rats were treated by osmotic minipump for 21 d with the selective serotonin reuptake inhibitors (SSRIs) paroxetine or sertraline, the selective norepinephrine reuptake inhibitor desipramine (DMI), or the monoamine oxidase inhibitor phenelzine. High-speed in vivo electrochemical recordings were used to assess the ability of the SSRI fluvoxamine to modulate the clearance of locally applied serotonin in the CA3 region of hippocampus in drug- or vehicle-treated rats. Fluvoxamine decreased the clearance of serotonin in rats treated with vehicle, DMI, or phenelzine but had no effect on the clearance of serotonin in SSRI-treated rats. SERT density in the CA3 region of the hippocampus of the same rats, assessed by quantitative autoradiography with tritiated cyanoimipramine ([(3)H]CN-IMI), was decreased by 80-90% in SSRI-treated rats but not in those treated with phenelzine or DMI. The serotonin content of the hippocampus was unaffected by paroxetine or sertraline treatment, ruling out neurotoxicity as a possible explanation for the SSRI-induced decrease in SERT binding and alteration in 5-HT clearance. Levels of mRNA for the SERT in the raphe nucleus were also unaltered by chronic paroxetine treatment. Based on these results, it appears that the SERT is downregulated by chronic administration of SSRIs but not other types of antidepressants; furthermore, the downregulation is not caused by decreases in SERT gene expression.

Transient expression of a functional serotonin transporter in Merkel cells during late gestation and early postnatal rat development.

We and others have previously identified serotonin transporter mRNA throughout the trigeminal system in the whisker region, trigeminal ganglion, trigeminal nucleus and thalamic relay stations. In order to further implicate a role for the serotonin transporter in this sensory system, we have now characterized serotonin transporter gene expression and function in primary cultures from the rat snout, at several stages of gestation. In this study, we have demonstrated a transient expression of serotonin transporter mRNA in quinacrine-positive Merkel cells between embryonic day 16 and postnatal day 5. Peak levels of mRNA occurred at embryonic day 20 and postnatal day 1. Merkel cells in culture exhibited a transient, antidepressant-sensitive [3H]-serotonin uptake, which was maximal at a time in culture corresponding to embryonic day 22 (day of birth). This transient uptake of serotonin suggests a role for this monoamine during a critical time period of the developing trigeminal sensory system. Regulation of extracellular serotonin levels by transporter activity may reflect the specific formation of the merkel cell-sensory neuron complex in an analogous mechanism by which serotonin modulates synaptogenesis in the central nervous system.

Effects of tamoxifen on serotonin transporter and 5-hydroxytryptamine 2A receptor binding sites and mRNA levels in the brain of ovariectomized rats with or without acute estradiol replacement

Estradiol-17β (E 2), in its positive feedback mode for gonadotropin release in the female rat, induces expression of the genes for the 5-hydroxytryptamine 2A receptor (5-HT 2AR) and the serotonin transporter (SERT) in the dorsal raphe nucleus (DRN) with a concomitant increase in the densities of 5-HT 2AR and the SERT in rat forebrain. The forebrain regions affected are those which, in humans, are concerned with the control of mood, mental state, cognition and emotion. Here we have used the mixed estradiol agonist/antagonist, tamoxifen, to determine whether this action of estradiol is mediated by cytoplasmic estradiol receptors. Acute treatment (∼32 h) of ovariectomized rats with estradiol benzoate (EB) increased significantly the amount of 5-HT 2AR mRNA and SERT mRNA in the DRN and the densities of 5-HT 2AR and SERT binding sites in the forebrain. These effects of EB were completely blocked by tamoxifen. Treatment with tamoxifen alone had no effect on either gene expression or the density of binding sites. Together, these data show that tamoxifen acts as a pure estradiol antagonist with respect to serotonergic mechanisms in brain. Detailed analysis of the effects of estradiol and tamoxifen on the DRN showed that SERT gene expression is constitutive only in the posterior DRN; in the anterior DRN, SERT gene expression appears to depend upon estrogen induction which is blocked by tamoxifen. Our findings strongly suggest that estradiol receptors are involved in mediating estradiol action on central serotonergic mechanisms and are relevant for our understanding of the effects of antiestrogens as well as estradiol on mood, mental state and cognition.

Androgen actions on central serotonin neurotransmission: relevance for mood, mental state and memory

Sex steroids exert potent effects on mood and mental state in the human. Our previous experimental findings in female rats suggest that these effects may be mediated, in part, by the action of estrogen on the 5-hydroxytryptamine 2A receptor (5-HT 2AR) and serotonin transporter (SERT) in brain. Here we review our recent findings on the effect of acute (∼32 h) testosterone manipulation on central 5-HT 2AR and SERT in male rats. Castration decreased while testosterone or estrogen, but not 5α-dihydrotestosterone (5α-DHT), increased significantly the content of 5-HT 2AR mRNA and SERT mRNA in the dorsal raphe nucleus (DR) and the density of 5-HT 2AR and SERT binding sites in higher centers of the brain. The lack of effect of 5α-DHT, a potent androgen which cannot be converted to estrogen, suggests that the action of testosterone depends upon its conversion to estrogen by aromatase. This may also explain why estrogen, but not testosterone or 5α-DHT, increased the density of 5-HT 2AR binding sites in the caudate-putamen, a brain region where aromatase is scarce. The estrogen induction of SERT mRNA is most prominent in the rostral DR and this together with the correlation between sensitivity of DR serotonin neurons to estrogen and neurotoxic amphetamine derivatives provides a potential topochemical handle with which to investigate testosterone/estrogen regulation of SERT gene expression. These findings are discussed in relation to the possible role of interactions between sex steroids and serotonin mechanisms in mood disorders, schizophrenia and Alzheimer's disease.

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

Serotonin transporter (SERT) mRNA and binding site densities in male rat brain affected by sex steroids

Estrogen increases serotonin transporter (SERT) mRNA and binding sites in female rat brain. In order to determine whether changes in SERT are gender- and steroid-specific we have now carried out studies on adult male Wistar rats which were either intact or castrated (under halothane anesthesia) and injected with arachis oil, estradiol benzoate (EB), testosterone propionate (TP) or the non-aromatizable androgen, 5α-dihydrotestosterone (5α-DHT). The number of SERT mRNA-expressing cells in the dorsal raphe (DR) nucleus was decreased by castration and increased by treatment (for ∼32 h) with EB or TP, but not 5α-DHT. Sex steroids had no effect on the number of SERT mRNA-expressing cells in the median raphe nucleus. The density of SERT sites, assessed by autoradiography of [ 3 H ]paroxetine binding, was significantly reduced in arcuate nucleus and median raphe after castration, and increased in arcuate, basolateral amygdala and ventromedial hypothalamic nucleus by treatment with EB or TP, but not 5α-DHT. Estradiol, but not testosterone or 5α-DHT reduced the density of SERT sites in midbrain central grey. These data show that testosterone as well as estrogen affects SERT expression in male brain, and that the action of testosterone probably depends upon its enzymatic conversion, by aromatase, to estradiol. Our findings may have implications for sex steroid control of mood and behavior, and the action of neurotoxic derivatives of amphetamine, such as 3,4-methylenedioxymethamphetamine, in the human.

Infraorbital nerve transection alters serotonin transporter expression in sensory pathways in early postnatal rat development

The serotonin transporter MRNA has been found throughout the trigeminal sensory system late in gestation and during early postnatal development, a period known to be critical for maturation of the sensory circuitry. The purpose of the present study was to determine whether sensory denervation in newborn rat pups would alter either the density or pattern of expression of the 5-HT transporter (5-HTT) within the trigeminal system. We combined autoradiographic localization of 5-HT transporters and in situ hybridization techniques to visualize both the transporter protein and mRNA in thalamic sensory neurons and in the somatosensory cortex following unilateral infraorbital nerve transection at postnatal day 1. For comparative purposes, similar measurements were conducted in thalamic visual neurons as well as in the visual cortex. Lesion of the infraorbital nerve decreased the [ 3H]citalopram labelling of 5-HT transporters in the ventral basal and ventral medial areas of the thalamus contralateral to the lesion, while labelling of 5-HT transporters was decreased in both contralateral and ipsilateral sides of the lateral genicuate (visual thalamus). Citalopram labelling of 5-HT transporters was not significantly altered in somatosensory or in cingulate cortex, however a significant decrease was observed in the visual cortex. In contrast, there were no obvious changes in the intensity of the 5-HT mRNA hybridization signal in sensory or visual thalamic areas. Given that the serotonin transporter regulates extracellular concentrations of 5-HT, the present data suggest that altered peripheral innervation and thereby altered sensory inputs to the thalamus during fetal development can potentially influence 5-HT transporter densities and thus, may influence extracellular levels of 5-HT in thalamus and cortex during a critical period of synapse formation. In turn, modulation of 5-HT transporter levels may influence extracellular concentrations of 5-HT in thalamus and cortex during a critical period of synapse formation.

Serotonin transporter on rat platelets: levels of mRNA underlie inherited differences in uptake kinetics

By the breeding selection for the extreme values of platelet serotonin transporter activity, two sublines of Wistar-derived rats, with constitutionally high or low platelet serotonin uptake (PSU), were previously developed. In order to study the genetic background of these inherited differences, comparative Northern blot analysis of the platelet serotonin transporter messenger RNA levels of the animals from the two sublines was performed. If the values of animals from the high-PSU subline are taken as 100%, animals from the low-PSU subline demonstrated lower values of both platelet serotonin uptake and transporter mRNA levels (amounting to 62 and 76% respectively). Correlation between platelet serotonin uptake and the respective levels of messenger RNA for the serotonin transporter ( r = 0.829, P < 0.01, N = 8) points to the same direction, indicating that the process of breeding selection for the extreme values of transporter kinetics has influenced transcription mechanisms of the serotonin transporter gene.

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.

Localization and Dynamic Regulation of Biogenic Amine Transporters in the Mammalian Central Nervous System

The monoamines, serotonin, dopamine, norepinephrine, epinephrine and histamine, play a critical role in the function of the hypothalamic–pituitary–adrenal axis and in the integration of information in sensory, limbic, and motor systems. The primary mechanism for termination of monoaminergic neurotransmission is through reuptake of released neurotransmitter by Na+,CI-dependent plasma membrane transporters. A second family of transporters packages monoamines into synaptic and secretory vesicles by exchange of protons. Identification of those cells which express these two families of neurotransmitter transporters is an initial step in understanding what adaptive strategies cells expressing monoamine transporters use to establish the appropriate level of transport activity and thus attain the appropriate efficiency of monoamine storage and clearance. The most recent advances in this field have yielded several surprises about their function, cellular and subcellular localization, and regulation, suggesting that these molecules are not static and most likely are the most important determinants of extracellular levels of monoamines. Here, information on the localization of mRNAs for these transporters in rodent and human brain is summarized along with immunohistochemical information at the light and electron microscopic levels. Regulation of transporters at the mRNA level by manipulation in rodents and differences in transporter site densities by tomographic techniques as an index of regulation in human disease and addictive states are also reviewed. These studies have highlighted the presence of monoamine neurotransmitter transporters in neurons but not in gliain situ.The norepinephrine transporter is present in all cells which are both tyrosine hydroxylase (TH)- and dopamine β-hydroxylase-positive but not in those cells which are TH- and phenyl-N-methyltransferase-positive, suggesting that epinephrine cells may have their own, unique transporter. In most dopaminergic cells, dopamine transporter mRNA completely overlaps with TH mRNA-positive neurons. However, there are areas in which there is a lack of one to one correspondence. The serotonin transporter (5-HTT) mRNA is found in all raphe nuclei and in the hypothalamic dorsomedial nucleus where the 5-HTT mRNA is dramatically reduced following immobilization stress. The vesicular monoamine transporter 2 (VMAT2) is present in all monoaminergic neurons including epinephrine- and histamine-synthesizing cells. Immunohistochemistry demonstrates that the plasma membrane transporters are present along axons, soma, and dendrites. Subcellular localization of DAT by electron microscopy suggests that these transporters are not at the synaptic density but are confined to perisynaptic areas, implying that dopamine diffuses away from the synapse and that contribution of diffusion to dopamine signalling may vary between brain regions. Interestingly, the presence of VMAT2 in vesicles underlying dendrites, axons, and soma suggests that monoamines may be released at these cellular domains. An understanding of the regulation of transporter function may have important therapeutic consequences for neuroendocrine function in stress and psychiatric disorders.

Long-term effects on serotonin transporter mRNA expression of chronic neonatal exposure to a serotonin reuptake inhibitor

Chronic administration of clomipramine or other serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitors to neonatal rats produces behaviours that resemble a depressive state in the adult animal, and this model is therefore regarded as a putative animal model of depression. Alterations in the activity of the central 5-HT system are important in understanding the pathophysiology of depression, and therefore, we examined whether this model was associated with changes in the expression of 5-HT 1A receptor, 5-HT 1B receptor, and 5-HT transporter mRNA in the dorsal raphe nucleus and the hippocampus. Wistar rats were injected twice daily with the serotonin reuptake inhibitors clomipramine and 5-chloro-1-[3-(dimethylamino)propyl]-1-(4-fluoro-phenyl)-1,3-dihydroiso-benzofurane, hydrochloride (code Lu 10-134-C) at doses of 15 mg kg −1 or vehicle i.p. from postnatal day 8 for 14 days. Groups of rats ( n=10) were either killed the day after the last injection or left undisturbed for 69 days before they were killed. The expression of 5-HT transporter, 5-HT 1A receptor, and 5-HT 1B receptor mRNA was examined in the dorsal raphe nucleus and in the CA1 of the hippocampus by means of quantitative in situ hybridisation histochemistry. Both compounds resulted in an increase in 5-HT transporter mRNA expression (40% more than vehicle) in the dorsal raphe nucleus the day after the last injection (postnatal day 22). A small but significant increase in 5-HT 1B receptor mRNA expression in the CA1 was seen after clomipramine, but not after Lu 10-134-C, probably reflecting clomipramine's affinity for both the 5-HT and noradrenaline transporters as well as for a number of monoamine receptor sites. Levels of 5-HT 1A receptor mRNA were unchanged. In contrast, 5-HT transporter mRNA expression in the dorsal raphe nucleus was significantly decreased in the adult after neonatal treatment with either of the two drugs compared to vehicle. No changes in 5-HT 1A receptor and 5-HT 1B receptor mRNA expression were observed in any of the regions examined in these animals. The results show that the persistent depressive behaviour previously shown in this model is also associated with changes in the expression of 5-HT transporter mRNA. This long-term alteration in gene expression may result from disturbances in 5-HT neurotransmission in the brain of the neonatal animals.

Serotonin transporters in adult rat brain astrocytes revealed by [3H]5-HT uptake into glial plasmalemmal vesicles

Cultured astrocytes derived from neonatal rat brain exhibited high affinity, Na+-dependent, paroxetine and fluoxetine sensitive [3H] 5-HT uptake. Reverse transcriptase-PCR demonstrated that astrocytes in culture expressed messenger RNA for the cloned serotonin transporter protein which has been characterised as the neuronal serotonin transporter. Although the serotonin transporter in cultured astrocytes displayed a Km value approximately 10 times greater than found in adult brain synaptosomes, these observations indicated that astrocytes in vitro may express the same serotonin transporter as neurons. Reverse transcriptase-PCR demonstrated the presence of serotonin transporter mRNA in the adult rat cerebral cortex, suggesting that astrocytes in vivo may express low levels of this mRNA. To investigate whether astrocytes in the adult CNS express functional serotonin transporters, glial plasmalemmal vesicles were prepared from cerebral cortex, representing a subcellular fraction composed primarily of vesicles derived from astrocytes. These vesicles were characterised by [3H]-glutamate and [3H]-dopamine uptake and by immunoblot analysis, using glial and synaptic markers: glutamate synthase, SNAP-25 and synaptobrevin. [3H]5-HT was taken up into glial plasmalemmal vesicles in a high affinity (Km approximately 40 nM), Na + dependent, paroxetine-sensitive manner. The [3H]5-HT uptake capacity (Vmax ) in these vesicles was approximately one quarter of that observed in synaptosomes. These data indicate that astrocytes in culture and in vivo are capable of 5-HT uptake via the previously characterised ‘neuronal’ serotonin transporter.