Evidence that serotonin reuptake modulators increase the density of serotonin innervation in the forebrain

The mechanism of action of commonly used antidepressants remains an issue of debate. In the experiments reported here we studied the effects of three representative compounds, the selective serotonin reuptake inhibitor fluoxetine, the selective serotonin reuptake enhancer tianeptine and the selective norepinephrine reuptake inhibitor desipramine on the structure of central serotonin pathways after a 4-week administration. We found that the serotonin modulators fluoxetine and tianeptine, but not desipramine, increase the density of 5-HT and serotonin transporter (SERT)-immunoreactive axons in the neocortical layer IV and certain forebrain limbic areas, such as piriform cortex and the shell region of nucleus accumbens. These changes were noted in the absence of a significant effect of serotonin antidepressants on the expression of tryptophan hydroxylase (TPH-2), i.e. the rate-limiting enzyme for 5-HT biosynthesis and of SERT at the mRNA level. In addition, we found that anterogradely filled terminal axons from injections of biotinylated dextran amine into the dorsal raphe showed significantly more branching in animals treated with fluoxetine compared with animals treated with liposyn vehicle. Our findings suggest that antidepressants may exert very selective structural effects on their cognate monoamine systems in normal animals and raise the possibility that neurotrophic mechanisms may play a role in their clinical efficacy.     

Serotonergic effects on feeding, but not hypothalamus-pituitary-adrenal secretion, are altered in ovine pregnancy

In ovine pregnancy, as in human pregnancy, hypothalamus-pituitary-adrenal activity is chronically increased. These studies were designed to test the hypotheses that expression of serotonergic genes and responsiveness to serotonin are increased in pregnancy. We tested the stimulatory effect of an acute, intracerebroventricular injection of the serotonin reuptake inhibitor fluoxetine on plasma ACTH and cortisol in ewes during late pregnancy or postpartum. We also tested the effect of lower-dose, longer-term stimulation by intracerebroventricular infusion of fluoxetine in pregnant and nonpregnant ewes over 6 days. Overall, we found that the stimulatory effect of fluoxetine on ACTH and cortisol was not significantly different between late-gestation and nonpregnant ewes, although the effect of acute fluoxetine administration was inversely related to plasma progesterone concentrations. Also, there were no differences in hypothalamic expression of the glucocorticoid and mineralocorticoid receptors, corticotropin-releasing hormone, AVP, the serotonin reuptake transporter, or the serotonin [5-hydroxytryptamine (5-HT)] receptors 5-HT(1A) and 5-HT(2A) with pregnancy or fluoxetine treatment. However, chronic fluoxetine infusion reduced food intake in the nonpregnant, but not pregnant, ewes. Expression of proopiomelanocortin mRNA in the hypothalamus was reduced in pregnant compared with nonpregnant ewes. Our results indicate that pregnancy does not increase responsiveness of ACTH and cortisol to serotonergic stimulation but, rather, that progesterone reduces the ACTH response. In addition, we found a reduced ability of serotonin to inhibit feeding in the pregnant ewes, consistent with a reduction in anorexic mechanisms in the pregnant state.     

Antihypertensive effects of fluoxetine and L-5-hydroxytryptophan in rats.

The combination of fluoxetine (10 mg/kg) and L-5-hydroxytryptophan (5-HTP) (10 mg/kg) significantly lowered blood pressure in spontaneously hypertensive rats and in rats made hypertensive by treatment with deoxycorticosterone (DOCA) and saline. Fluoxetine alone also had a significant effect on blood pressure in DOCA hypertensive rats, but not as great an effect as the combination. Since fluoxetine is an inhibitor of serotonin reuptake and 5-HTP is the serotonin precursor, the antihypertensive effect of this drug combination strengthens previous evidence that serotonin neurons have a role in the central regulation of blood pressure.     

Relation between brain 5-HIAA levels and the release of serotonin into brain synapses

Our findings in experiments using reserpine, an amine releaser, and fluoxetine, a serotonin uptake blocker, indicate that the reuptake of serotonin from brain synapses precedes its transformation to 5-hydroxyindoleacetic acid (5-HIAA). Male rats were injected with reserpine or fluoxetine alone, or with fluoxetine one hour before reserpine; control animals received diluents. Reserpine lowered brain serotonin and raised brain 5-HIAA levels. Fluoxetine alone did not change serotonin levels but lowered 5-HIAA. Fluoxetine completely antagonized the reserpine-induced increase in 5-HIAA, and significantly enhanced its depletion of serotonin. In order to determine whether the ability of fluoxetine to block the rise in 5-HIAA after reserpine resulted from its effect on serotonin reuptake or from suppression of impulse flow along serotoninergic neurons, we also examined the effects of the drugs on serotonin metabolism in distal portions of acutely transected neurons (which, presumably, were no longer able to conduct impulses). No differences were noted between the responses of intact and lesioned serotoninergic neurons, indicating that fluoxetine's blockade of the rise in brain 5-HIAA results from its effect on serotonin reuptake.     

GluN2B-containing NMDA receptors as possible targets for the neuroprotective and antidepressant effects of fluoxetine

Accumulating evidence has indicated the involvement of glutamatergic neurotransmission in the pathophysiology of excitotoxicity and in the mechanism of action of antidepressants. We have previously shown that tricyclic desipramine and the selective serotonin reuptake inhibitor fluoxetine inhibit NMDA receptors (NMDARs) in the clinically relevant, low micromolar concentration range. As the different subtypes of NMDARs are markedly different in their physiological and pathological functions, our aim was to investigate whether the effect of antidepressants is subtype-specific. Using whole-cell patch-clamp recordings in rat cortical cell cultures, we studied the age-dependence of inhibition of NMDA-induced currents after treatment with desipramine and fluoxetine, as the expression profile of the NMDAR subtypes changes as a function of days in vitro. We also investigated the inhibitory effect of these antidepressants on NMDA-induced currents in HEK 293 cell lines that stably expressed rat recombinant NMDARs with GluN1a/GluN2A or GluN1a/GluN2B subunit compositions. The inhibitory effect of desipramine was not age-dependent, whereas fluoxetine displayed a continuously decreasing inhibitory profile, which was similar to the GluN1/GluN2B subtype-selective antagonist ifenprodil. In HEK 293 cells, desipramine equally inhibited NMDA currents in both cell lines, whereas fluoxetine showed an inhibitory effect only in cells that expressed the GluN1/GluN2B subtype. Our data show that fluoxetine is a selective inhibitor of GluN2B-containing NMDARs, whereas desipramine inhibits both GluN1/GluN2A and GluN1/GluN2B subtypes. As the clinical efficacy of these drugs is very similar, the putative NMDAR-associated therapeutic effect of antidepressants may be mediated only via inhibition of the GluN2B-containing subtype. The manifestation of the GluN1/GluN2B-selectivity of fluoxetine suggests the neuroprotective potential for this drug in both acute and chronic neurodegenerative disorders.     

Effect of long-term fluoxetine treatment on the human serotonin transporter in Caco-2 cells

Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) broadly used in the treatment of human mood disorders and gastrointestinal diseases involving the serotoninergic system. The effectiveness of this therapy depends on repeated long-term treatment. Most of the long-term studies in vivo of SSRI effects on serotoninergic activity have focused on their effects on autoreceptors or postsynaptic receptors. The chronic effect of SSRIs on the activity of the serotonin transporter (SERT) has been less studied and the results have been contradictory. The aim of this study was to determine the specific effect of long-term fluoxetine treatment on human serotonin transporter (hSERT) in vitro, by using the human enterocyte-like cell line Caco-2. Results show that fluoxetine diminished the 5-HT uptake in a concentration-dependent way and that this effect was reversible. Fluoxetine affected mainly the hSERT transport rate by reducing the availability of the transporter in the membrane with no significant alteration of either the total hSERT protein content or the hSERT mRNA level. These results suggest that the effect of fluoxetine on the expression of hSERT is post-translational and has shown itself to be independent of PKC and PKA activity. This study may be useful to clarify the effect of the long-term fluoxetine therapy in both gastrointestinal and central nervous system disorders.     

Spatial-specific action of serotonin within the leech midbody ganglion

Serotonin is a conspicuous neuromodulator in the nervous system of many vertebrates and invertebrates. In previous experiments performed in the leech nervous system, we compared the effect of the amine released from endogenous sources [using selective serotonin reuptake inhibitors (SSRIs), e.g. fluoxetine] with that of bath-applied serotonin. The results suggested that the amine does not reach all its targets in a uniform way, but produces the activation of an interneuronal pathway that generated specific synaptic responses on different neurons. Taking into account that the release of the amine is often regulated at the presynaptic level, we have investigated whether autoreceptor antagonists mimic the SSRIs effect. We found that methiothepin (100 microM) produced similar effects than fluoxetine. To further test the hypothesis that endogenous serotonin produce its effect by acting locally at specific sites, we analyzed the effect of iontophoretic applications of serotonin. We found a site in the neuropil of the leech ganglia where serotonin application mimicked the effect of the SSRIs and the 5-HT antagonist. The results further support the view that the effect of serotonin exhibits a spatial specificity that can be relevant to understand its modulatory actions.     

Pharmacologic evidence for a serotonin neural pathway involved in hypothalamus-pituitary-adrenal function in rats.

The hypothesis that a serotonin neural pathway stimulates ACTH secretion in rats was supported by pharmacologic data. Fluoxetine, an inhibitor of serotonin reuptake, caused a dose-related elevation of plasma corticosterone levels in intact but not in hypophysectomized rats. The previously-reported elevation of plasma corticosterone by 5-hydroxytryptophan (5HTP) was confirmed and shown to be stereospecific, L-5HTP being much more active than D-5HTP. Simultaneous injection of subeffective doses of fluoxetine and L-5HTP caused marked elevation of plasma corticosterone. Fluoxetine pretreatment potentiated the elevation of plasma corticosterone by L-5HTP. Although the elevation of plasma corticosterone by fluoxetine was of short duration (perhaps due to compensatory reduction of serotonin release), the potentiation of the L-5HTP effect by fluoxetine lasted for more than 24 hrs as predicted by the duration of uptake inhibition by fluoxetine. The dose-response characteristics for corticosterone elevation and L-5HTP potentiation by fluoxetine were similar to those for serotonin uptake blockade.     

Decreased plasma leptin levels in lean and obese Zucker rats after treatment with the serotonin reuptake inhibitor fluoxetine.

Leptin inhibits feeding, stimulates thermogenesis and decreases body weight. Serotonin reduces food intake when injected into the hypothalamus and may interact with other neurotransmitters in the control of appetite. We therefore investigated the effects of the serotonergic drug fluoxetine, which inhibits serotonin reuptake, on food intake and plasma leptin levels in lean and obese Zucker rats. Fluoxetine significantly reduced food intake in lean and obese rats, both acutely after a single injection (10 mg/kg) and during continuous subcutaneous infusion (10 mg/kg/day for 7 days). Plasma leptin levels were reduced after both 4 hours and 7 days of fluoxetine administration in lean and after 7 days in fatty rats (all p<0.01). We have previously suggested that serotonin may decrease food intake by inhibiting neuropeptide Y neurones, and we further suggest that it also inhibits leptin, possibly by an effect on white adipose tissue.     

Modification of antiallodynic and antinociceptive effects of morphine by peripheral and central action of fluoxetine in a neuropathic mice model

We have previously reported that serotonin concentration was reduced in the brain of mice with neuropathic pain and that it may be related to reduction of morphine analgesic effects. To further prove this pharmacological action, we applied fluoxetine, a selective serotonin reuptake inhibitor, to determine whether it suppressed neuropathic pain and examined how its different administration routes would affect antinociceptive and antiallodynic effects of morphine in diabetic (DM) and sciatic nerve ligation (SL) mice, as models of neuropathic pain. Antiallodynia and antinociceptive effect of drugs were measured by using von Frey filament and tail pinch tests, respectively. Fluoxetine given alone, intracerebroventicularly (i.c.v., 15 microg/mouse) or intraperitoneally (i.p., 5 and 10 mg/kg) did not produce any effect in either model. However, fluoxetine given i.p. enhanced both antiallodynic and antinociceptive effects of morphine. Administration of fluoxetine i.c.v., slightly enhanced only the antiallodynic effect of morphine in SL mice. Ketanserine, a serotonin 2A receptor antagonist (i.p., 1 mg/kg) and naloxone, an opioid receptor antagonist (i.p., 3 mg/kg), blocked the combined antinociceptive effect of fluoxetine and morphine. Our data show that fluoxetine itself lacks antinociceptive properties in the two neuropathy models, but it enhances the analgesic effect of morphine in the periphery and suggests that co-administration of morphine with fluoxetine may have therapeutic potential in treatment of neuropathic pain.     

Fluoxetine treatment increases trabecular bone formation in mice

Mounting evidence exists for the operation of a functional serotonin (5-HT) system in osteoclasts and osteoblasts, which involves both receptor activation and 5-HT reuptake. In previous work we showed that the serotonin transporter (5-HTT) is expressed in osteoclasts and that its activity is required by for osteoclast differentiation in vitro. The purpose of the current study was to determine the effect of treatment with fluoxetine, a specific serotonin reuptake inhibitor, on bone metabolism in vivo. Systemic administration of fluoxetine to Swiss-Webster mice for 6 weeks resulted in increased trabecular BV and BV/TV in femurs and vertebrae as determined by micro-computed tomography (microCT). This correlated with an increase in trabecular number, connectivity, and decreased trabecular spacing. Fluoxetine treatment also resulted in increased volume in vertebral trabecular bone. However, fluoxetine-treated mice were not protected against bone loss after ovariectomy, suggesting that its anabolic effect requires the presence of estrogen. The effect of blocking the 5-HTT on bone loss following an LPS-mediated inflammatory challenge was also investigated. Subcutaneous injections of LPS over the calvariae of Swiss-Webster mice for 5 days resulted in increased numbers of osteoclasts and net bone loss, whereas new bone formation and a net gain in bone mass was seen when LPS was given together with fluoxetine. We conclude that fluoxetine treatment in vivo leads to increased bone mass under normal physiologic or inflammatory conditions, but does not prevent bone loss associated with estrogen deficiency. These data suggest that commonly used anti-depressive agents may affect bone mass.     

Serotonin (5HT), fluoxetine, imipramine and dopamine target distinct 5HT receptor signaling to modulate Caenorhabditis elegans egg-laying behavior

Drugs that target the serotonergic system are the most commonly prescribed therapeutic agents and are used for treatment of a wide range of behavioral and neurological disorders. However, the mechanism of the drug action remain a conjecture. Here, we dissect the genetic targets of serotonin (5HT), the selective 5HT reuptake inhibitor (SSRI) fluoxetine (Prozac), the tricyclic antidepressant imipramine, and dopamine. Using the well-established serotonergic response in C. elegans egg-laying behavior as a paradigm, we show that action of fluoxetine and imipramine at the 5HT reuptake transporter (SERT) and at 5HT receptors are separable mechanisms. Even mutants completely lacking 5HT or SERT can partially respond to fluoxetine and imipramine. Furthermore, distinct mechanisms for each drug can be recognized to mediate these responses. Deletion of SER-1, a 5HT1 receptor, abolishes the response to 5HT but has only a minor effect on the response to imipramine and no effect on the response to fluoxetine. In contrast, deletion of SER-4, a 5HT2 receptor, confers significant resistance to imipramine while leaving the responses to 5HT or fluoxetine intact. Further, fluoxetine can stimulate egg laying via the Gq protein EGL-30, independent of SER-1, SER-4, or 5HT. We also show that dopamine antagonizes the 5HT action via the 5HT-gated ion channel MOD-1 signaling, suggesting that this channel activity couples 5HT and dopamine signaling. These results suggest that the actions of these drugs at specific receptor subtypes could determine their therapeutic efficacy. SSRIs and tricyclic antidepressants may regulate 5HT outputs independently of synaptic levels of 5HT.     

Functional significance of glycogen synthase kinase-3 regulation by serotonin

Serotonin modulates brain physiology and behavior and has major roles in brain diseases involving abnormal mood and cognition. Enhancing brain serotonin has been found to regulate glycogen synthase Kinase-3 (GSK3), but the signaling mechanism and functional significance of this regulation remain to be determined. In this study, we tested the signaling mechanism mediating 5-HT1A receptor-regulated GSK3 in the hippocampus. Using mutant GSK3 knock-in mice, we also tested the role of GSK3 in the behavioral effects of 5-HT1A receptors and the serotonin reuptake inhibitor fluoxetine. The results showed that activation of 5-HT1A receptors by 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) increased phosphorylation of the N-terminal serine of both GSK3α and GSK3β in several areas of the hippocampus. The effect of 8-OH-DPAT was accompanied by an increase in the active phosphorylation of Akt, and was blocked by LY294002, an inhibitor of phosphoinositide 3-kinases (PI3K). Phosphorylation of GSK3β, but not GSK3α, was necessary for 5-HT1A receptors to suppress the hippocampus-associated contextual fear learning. Furthermore, acute fluoxetine treatment up-regulated both phospho-Ser21-GSK3α and phospho-Ser9-GSK3β in the hippocampus. Blocking phosphorylation of GSK3α and GSK3β diminished the anti-immobility effect of fluoxetine treatment in the forced swim test, wherein the effect of GSK3β was more prominent. These results together suggest that PI3K/Akt is a signaling mechanism mediating the GSK3-regulating effect of 5-HT1A receptors in the hippocampus, and regulation of GSK3 is an important intermediate signaling process in the behavioral functions of 5-HT1A receptors and fluoxetine.     

The selective serotonin reuptake inhibitor fluoxetine increases spontaneous afferent firing,but not mechanonociceptive sensitization,in octopus

Serotonin is a widely studied modulator of neural plasticity. Here we investigate the effect of fluoxetine, a selective serotonin reuptake inhibitor, on short-term, peripheral nociceptive plasticity in the neurologically complex invertebrate, octopus. After crush injury to isolated mantle (body wall) tissue, application of 10 nM fluoxetine increased spontaneous firing in crushed preparations, but had a minimal effect on mechanosensory sensitization. Effects largely did not persist after washout. We suggest that transiently elevated, endogenous serotonin may help promote initiation of longer-term plasticity of nociceptive afferents and drive immediate and spontaneous behaviors aimed at protecting wounds and escaping dangerous situations.     

Fish on Prozac: a simple, noninvasive physiology laboratory investigating the mechanisms of aggressive behavior in Betta splendens

The neuromodulator serotonin is an important regulator of aggressive behavior in vertebrates. Experimentally increasing synaptic levels of serotonin with fluoxetine, a selective serotonin reuptake inhibitor, has been shown to reliably decrease the expression of aggressive behavior. Here, we describe a method by which fluoxetine can be noninvasively administered to male Betta splendens (an attractive model for the study of aggressive behavior) and describe a simple laboratory exercise that allows students to experimentally investigate the physiological mechanisms of aggressive behavior. We demonstrate that relatively short-term exposure (3 h) of male bettas to as little as 3 microg/ml of fluoxetine-treated aquarium water is sufficient to reduce the expression of specific aggressive behaviors. We emphasize the physiological concepts that can be addressed with this exercise, including the role of the serotonergic system in regulating aggression, and the interplay of environmental contaminants and physiology in regulating the expression of behavior. We also highlight important aspects of experimental design. This exercise can be flexibly altered to accommodate one or several laboratory periods. It is also low cost, is low impact to the animals, and requires minimal preparation time for instructors.     

Effect of serotonin-acting agents on the serotonin content of immune cells. A peculiar observation

The effect of the tryptophan hydroxylase inhibitor, PCPA methylester, the serotonin reuptake inhibitor fluoxetine and MAO-A inhibitor clorgyline on the serotonin content of rat immune cells was studied, using labelled antibodies and flow cytometry. Each molecule significantly increased in males the serotonin concentration of peritoneal lymphocytes and the monocyte-macrophage-granulocyte group (mo-gran), however the agents were ineffective towards mast cells. In females fluoxetine and clorgyline increased the serotonin concentration in peritoneal lymphocytes and mo-gran. Fluoxetine also increased the serotonin level in mast cells. Thymus was absolutely resistant to the drugs in both genders. The results call attention (1) to the reverse effect of serotonin-acting agents on immune cells, (2) to the influence of the milieu where the cell is located and (3) the effect of gender.     

Metabolism and Pharmacokinetics of Selective Serotonin Reuptake Inhibitors

1. Five drugs with the predominant pharmacologic effect of inhibiting the neuronal reuptake of serotonin are available worldwide for clinical use. This class of psychoactive drugs, known as selective serotonin reuptake inhibitors (SSRIs), is comprised of fluoxetine, sertraline, paroxetine, fluvoxamine, and citalopram.2. The SSRIs appear to share similar pharmacodynamic properties which translate to efficacy in the treatment of depression and anxiety syndromes. The drugs are differentiated by their pharmacokinetic properties with regard to stereochemistry, metabolism, inhibition of cytochrome enzymes, and participation in drug–drug interactions. Studies focusing on the relationship of plasma drug concentration to therapeutic and adverse effects have not confirmed the value of plasma concentration monitoring.3. This review summarizes the metabolism and relevant pharmacokinetic properties of the SSRIs.     

The acute effects of 5HTP, fluoxetine and quipazine on insulin and glucagon release in the intact rat.

5-hydroxytryptophan (5HTP), the immediate precursor of serotonin, induces a release of insulin and glucagon in the intact rat. These effects of 5HTP, which have previously been shown to be blocked by L-aromatic amino acid decarboxylase inhibition, were also prevented by methysergide (a serotonin receptor antagonist). Quipazine (a serotonin receptor agonist) did not alter pancreatic hormone release. Fluoxetine, a serotonin neuronal reuptake blocker did not effect insulin secretion and had a slight glucagon stimulatory effect, however the effects of 5HTP on insulin and glucagon release were not potentiated by fluoxetine pretreatment. Alpha and beta-adrenergic receptor blockade did not alter the pancreatic effects of 5HTP.     

Fluoxetine and nortriptyline affect NTPDase and 5'-nucleotidase activities in rat blood serum

Depression is a serious condition associated with considerable morbidity and mortality. Selective serotonin reuptake inhibitors and tricyclic antidepressants, such as fluoxetine and nortriptyline, respectively, were commonly used in treatment for depression. Selective serotonin reuptake inhibitors have been associated with increased risk of bleeding complications, possibly as a result of inhibition of platelet aggregation. ATP, ADP and adenosine are signaling molecules in the vascular system and nucleotidases activities are considered an important thromboregulatory system which functions in the maintenance of blood fluidity. Therefore, here we investigate the effect of in vivo (acute and chronic) and in vitro treatments with the antidepressant drugs on nucleotidases activities in rat blood serum. In acute treatment, nortriptyline decreased ATP hydrolysis (41%), but not altered ADP and AMP hydrolysis. In contrast, fluoxetine did not alter NTPDase and ecto-5'-nucleotidase activities. A significant inhibition of ATP, ADP, and AMP hydrolysis were observed in chronic treatment with fluoxetine (60%, 32%, and 42% for ATP, ADP, and AMP hydrolysis, respectively). Similar effects were shown in chronic treatment with nortriptyline (37%, 41%, and 30% for ATP, ADP, and AMP hydrolysis, respectively). In addition, there were no significant changes in NTPDase and ecto-5'-nucleotidase activities when fluoxetine and nortriptyline (100, 250, and 500 microM) were tested in vitro. Our results have shown that fluoxetine and nortriptyline changed the nucleotide catabolism, suggesting that homeostasis of vascular system can be altered by antidepressant treatments.