The influence of dehydroepiandrosterone and 8-OH-DPAT on the caloric intake and hypothalamic neurotransmitters of lean and obese Zucker rats

The 5 HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetraline (8-OH-DPAT) increases the food intake of satiated Zucker rats, both lean and obese. Associated with this increased intake are changes in the hypothalamic content of serotonin and its metabolite, 5-HIAA (5-hydroxyindole-3-acetic acid); serotonin is increased while the level of 5-HIAA is decreased. Analysis of individual 5-HIAA/5-hydroxytryptamine (5-HT) ratios, a measure of serotonin turnover indicate that 8-OH DPAT affected serotonin turnover equally and dramatically in both phenotypes. This would be an expected physiological action of an autofeedback mechanism by a 5-HT(1A) receptor agonist. Dehydroepiandrosterone (DHEA) at doses as low as 10 mg/kg blocks the 8-OH-DPAT-induced increase in food intake but does not alter food intake of control satiated Zucker rats. The mechanism of DHEA's action was investigated by monitoring the steroid's effect on hypothalamic neurotransmitters in this satiated model. DHEA by itself induced some change in 5-HIAA in the obese satiated model but not the lean. 8-OH-DPAT, by itself, dramatically decreased serotonin turnover in either lean or obese rats, and DHEA combined with 8-OH-DPAT did not further change serotonin turnover, suggesting DHEA may work through mechanisms other than monoamines to cause its inhibition of 8-OH-DPAT-induced behavioral effects at such low doses.     

Serotonin and fluoxetine modulate bone cell function in vitro

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

Influence of Repeated Levodopa Administration on Rabbit Striatal Serotonin Metabolism,and Comparison Between Striatal and CSF Alterations

Parkinson's disease (PD) is characterized by decreased striatal dopamine, but serotonin (5-HT) is also reduced. Because 5-HT decreases following a single levodopa injection, levodopa has been suggested to contribute to PD's serotonergic deficits. However, in a recent study, rat striatal serotonin levels were reported to increase following 15-day levodopa administration. To address this issue, we administered levodopa (50 mg/kg) to rabbits for 5 days, then measured serotonin, its precursors tryptophan and 5-hydroxytryptophan (5-HTP), and its major metabolite 5-hydroxyindole-acetic acid (5-HIAA) in striatum and CSF. Striatal serotonin and tryptophan were unchanged, while 5-HTP and 5-HIAA increased 4- and 7-fold, respectively. CSF 5-HTP and 5-HIAA were also significantly increased. In levodopa-treated animals, 5-HTP concentrations were moderately correlated (r = 0.679) between striatum and CSF, while weak correlations were present between striatal and CSF concentrations of both serotonin and 5-HIAA. These results suggest that repeated levodopa treatment increases striatal serotonin turnover without changing serotonin content. However, levodopa-induced alterations in striatal serotonin metabolism may not be accurately reflected by measurement of serotonin and 5-HIAA in CSF.     

Synthesis and characterization of photolabile derivatives of serotonin for chemical kinetic investigations of the serotonin 5-HT(3) receptor

A series of photolabile o-nitrobenzyl derivatives of serotonin (caged serotonin) were synthesized: the amine-linked serotonin derivatives N-(2-nitrobenzyl) serotonin (Bz-5HT) and N-(alpha-carboxy-2-nitrobenzyl) serotonin (N-CNB-5HT), and O-alpha-carboxy-2-nitrobenzyl) serotonin (O-CNB-5HT), which has the caging group attached to the phenolic OH group. All the derivatives released free serotonin when excited by 308-nm or 337-nm laser pulses. The time constant of serotonin release from N-CNB-5HT was 1. 2 ms, with a quantum yield of 0.08. This is too slow for rapid chemical kinetic measurements. O-CNB-5HT is suitable for transient kinetic investigations of the serotonin 5-HT(3) receptor. It released serotonin with a time constant of 16 micros and a quantum yield of 0.03. The biological properties of O-CNB-5HT were evaluated, and the applicability of the compound for kinetic studies of the 5-HT(3) receptor was demonstrated. O-CNB-5HT does not activate the 5-HT(3) receptor by itself, nor does it modulate the response of a cell when co-applied with serotonin. When irradiated with a 337-nm laser pulse, O-CNB-5HT released free serotonin that evoked 5-HT(3) receptor-mediated whole-cell currents in NIE-115 mouse neuroblastoma cells.     

Synthesis and Pharmacology of the enantiomers of the potential atypical antipsychotic agents 5-OMe-BPAT and 5-OMe-(2,6-di-OMe)-BPAT.

The optically pure enantiomers of the potential atypical antipsychotic agents 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 5) and 5-methoxy-2-{N-[2-(2,6-dimethoxy)benzamidoethyl]-N-n-propylamino}t etralin [5-OMe-(2,6-di-OMe)-BPAT, 6] were synthesized and evaluated for their in vitro binding affinities at alpha1-, alpha2-, and beta-adrenergic, muscarinic, dopamine D1, D2A, and D3, and serotonin 5-HT1A and 5-HT2 receptors. In addition, their intrinsic efficacies at serotonin 5-HT1A receptors were established in vitro. (S)- and (R)-5 had high affinities for dopamine D2A, D3, and serotonin 5-HT1A receptors, moderate affinities for alpha1-adrenergic and serotonin 5-HT2 receptors, and no affinity (Ki > 1000 nM) for the other receptor subtypes. (S)- and (R)-6 had lower affinities for the dopamine D2A and the serotonin 5-HT1A receptor, compared to (S)- and (R)-5, and hence showed some selectivity for the dopamine D3 receptor. The interactions with the receptors were stereospecific, since the serotonin 5-HT1A receptor preferred the (S)-enantiomers, while the dopamine D2A and D3 receptors preferred the (R)-enantiomers of 5 and 6. The intrinsic efficacies at the serotonin 5-HT1A receptor were established by measuring their ability to inhibit VIP-induced cAMP production in GH4ZD10 cells expressing serotonin 5-HT1A receptors. Both enantiomers of 5 behaved as full serotonin 5-HT1A receptor agonists in this assay, while both enantiomers of 6 behaved as weak partial agonists. The potential antipsychotic properties of (S)- and (R)-5 were evaluated by establishing their ability to inhibit d-amphetamine-induced locomotor activity in rats, while their propensity to induce extrapyramidal side-effects (EPS) in man was evaluated by determining their ability to induce catalepsy in rats. Whereas (R)-5 was capable of blocking d-amphetamine-induced locomotor activity, indicative of dopamine D2 receptor antagonism, (S)-5 even enhanced the effect of d-amphetamine, suggesting that this compound has dopamine D2 receptor-stimulating properties. Since both enantiomers also were devoid of cataleptogenic activity, they are interesting candidates for further exploring the dopamine D2/serotonin 5-HT1A hypothesis of atypical antipsychotic drug action.     

Serotonin localization and its functional significance during mouse preimplantation embryo development

Serotonin is a neurotransmitter functioning also as a hormone and growth factor. To further investigate the biological role of serotonin during embryo development, we analysed serotonin localization as well as the expression of specific serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos. The functional significance of serotonin during the preimplantation period was examined by studying the effects of serotonin on mouse embryo development. Embryo exposure to serotonin (1 microM) highly significantly reduced the mean cell number, whereas lower concentrations of serotonin (0.1 microM and 0.01 microM) had no significant effects on embryo cell numbers. In all serotonin-treated groups a significant increase in the number of embryos with apoptotic and secondary necrotic nuclei was observed. Expression of serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos was confirmed by in situ hybridization showing a clearly distinct punctate signal. Immunocytochemistry results revealed the localization of serotonin in oocytes and embryos to the blastocyst stage as diffuse punctate cytoplasmic labelling. It appears that endogenous and/or exogenous serotonin in preimplantation embryos could be involved in complex autocrine/paracrine regulations of embryo development and embryo-maternal interactions.     

Relationship between serotonergic measures in periphery and the brain of mouse.

Circadian rhythm and the relationship between the concentration of serotonin (5HT) and related substances (5-hydroxyindoleacetic acid; 5HIAA and tryptophan; Trp) in mouse brain, stomach and blood have been studied. All factors underwent circadian changes in the brain and blood. 5HT and 5HIAA levels in the stomach showed no circadian fluctuation. The concentrations of 5HT in the brain and blood did not correlate. Significant correlations were found between other serotonergic parameters analyzed in brain, stomach and blood. A significant negative correlation was observed between brain 5HIAA and blood 5HIAA. The concentration of tryptophan in the brain was correlated with the plasma total tryptophan level. There was fairly significant correlation (p less than 0.06) between brain serotonin and plasma tryptophan levels. The brain serotonin and tryptophan levels were strongly correlated (R = 0.410, p less than 0.03). Significant negative correlation was found between serotonin in the blood and serotonin in the stomach as well as between its level in the brain and in the stomach. The significance of these findings and their relationship to the use of peripheral serotonergic system as a model of neurons are discussed.     

Zwitterionic and anionic forms of a serotonin analog as transport substrates

4,6-Difluoroserotonin, a serotonin analog with an acidic 5-hydroxyl proton (pK alpha = 7.97) relative to serotonin (pK alpha = 10.73), was tested as a substrate for the biogenic amine transporter of bovine chromaffin granules and the plasma membrane serotonin transporter of human blood platelets. The platelet serotonin transporter transports this analog with identical rates as those for serotonin, both at pH 6.7, where the hydroxyl group is predominantly protonated and at pH 9, where it is largely dissociated. In contrast, the chromaffin granule biogenic amine transporter prefers the form of 4,6-difluoroserotonin with a protonated 5-hydroxyl group. Thus, the KM for 4,6-difluoroserotonin increases, and Vmax decreases (relative to the values for serotonin) as the pH increases from 7 to 9. This effect may reflect a specific requirement for the protonated hydroxyl group in substrate translocation, as opposed to binding, since the KI for 4,6-difluoroserotonin inhibition of serotonin transport is the same as the KM for serotonin from pH 7 to 9.     

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

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

Serotonin systems in the inner and outer plexiform layers of the vertebrate retina

In retinas of certain nonmammalian vertebrate species such as frog, pigeon, and chick, serotonin appears to function as the neurotransmitter of a specific population of amacrine cells. Neurochemical and morphological studies have demonstrated high endogenous levels of 5-hydroxytryptamine (5-HT) as well as uptake, release, and receptor-binding activity restricted to the inner plexiform layer. In retinas from most mammalian species, uptake, release, and receptor-binding activity have also been localized to amacrine cell terminals in the inner plexiform layer. However, serotonin content in mammalian retinas is low, and attempts to localize the endogenous store of 5-HT have failed. Thus the status of serotonin as a candidate in mammalian retina is still open to question. Our more recent studies have revealed a light-sensitive serotonin system associated with photoreceptor terminals in retinas of Long-Evans rats. Uptake, synthesis, and release of [3H]serotonin have been demonstrated. Endogenous levels of 5-HT decrease in the dark and increase in the light. Electrophysiological studies are needed to illucidate the functional role(s) of serotonin within retinas of different species.     

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.     

Circadian and seasonal rhythms of 5-HT receptor subtypes, membrane anisotropy and 5-HT release in hippocampus and cortex of the rat.

Specific serotonin binding (5-HT1, 5-HT1A, and 5-HT2 subtypes) and membrane anisotropy were measured at 2 h intervals over a 24 h period in the hippocampus and cortex of Wistar WU rats, housed under a 12 h light-dark cycle, with lights on at 07.00. All experiments were performed both in March and December. In the hippocampus significant circadian rhythms could be ascertained for 5-HT1 binding sites in March and December while for 5-HT1A (subtype of 5-HT1) binding sites the circadian rhythm was only significant in March. The membrane anisotropy also showed significant variations only in March. Circadian rhythms were also found in the cortex for 5-HT1 (December) and 5-HT2 (March and December) binding sites as well as for the membrane anisotropy (December). A correlation was found between membrane anisotropy and 5-HT1 and 5-HT2 binding sites in hippocampus and cortex, respectively. A circadian rhythmicity was also observed for serotonin release as measured by in vivo voltammetry in both brain areas. The results obtained on the diurnal variations of serotonin receptor subtypes and serotonin release and the probable inverse relationship of these two parameters may be relevant in understanding the coupling of pre- and postsynaptic activity.     

Effects of short- and long-term neuroleptic treatment on brain serotonin synthesis and turnover: focus on the serotonin hypothesis of schizophrenia

Short-term (90 min) administration of haloperidol (2 mg/kg), or chlorpromazine (10 mg/kg) increased the activity of tryptophan hydroxylase as well as the levels of 5-hydroxytryptamine (serotonin) and 5-hydroxyindoleacetic acid in mid-brain of rats. The chronic neuroleptic treatment (21 days) produced more pronounced changes in all parameters related to serotonin synthesis and turnover. The activity of tryptophan hydroxylase in mid-brain was further augmented; the levels of 5-hydroxytryptamine and 5-hydroxyindole-acetic acid were significantly elevated not only in mid-brain, but also in several other discrete regions examined. These data suggest that neuroleptics enhance the synthesis and utilization of brain serotonin. The role of brain serotonergic neurons in the pathophysiology of schizophrenia is further considered.     

Measurement of antioxidant ability of melatonin and serotonin by the DMPD and CUPRAC methods as trolox equivalent

Melatonin (N-acetyl-5-methoxytryptamine) is the chief secretory product of the pineal gland and synthesized enzymatically from serotonin (5-hydroxytryptamine). These indoleamine derivatives play an important role in the prevention of oxidative damage. In the present study, DMPD radical scavenging and cupric ion (Cu(2+)) reducing ability of melatonin and serotonin as trolox equivalent antioxidant activity (TEAC) was investigated. Melatonin and serotonin demonstrated 73.5 and 127.4 microg/mL trolox equivalent DMPD( radical+) scavenging activity at the concentration of 100 microg/mL. Also, at the same concentration, melatonin and serotonin showed 14.41 and 116.09 microg/mL trolox equivalent cupric ion (Cu(2+)) reducing ability. These results showed that melatonin and serotonin had marked DMPD(radical+) radical scavenging and cupric ions (Cu(2+)) reducing ability. Especially, serotonin had higher DMPD radical scavenging and cupric ions (Cu(2+)) reducing activity than melatonin because of its phenolic group.     

A serotonin receptor antagonist induces oocyte maturation in both frogs and mice: evidence that the same G protein-coupled receptor is responsible for maintaining meiosis arrest in both species

Accumulating evidence has indicated that vertebrate oocytes are arrested at late prophase (G2 arrest) by a G protein coupled receptor (GpCR) that activates adenylyl cyclases. However, the identity of this GpCR or its regulation in G2 oocytes is unknown. We demonstrated that ritanserin (RIT), a potent antagonist of serotonin receptors 5-HT2R and 5-HT7R, released G2 arrest in denuded frog oocytes, as well as in follicle-enclosed mouse oocytes. In contrast to RIT, several other serotonin receptor antagonists (mesulergine, methiothepine, and risperidone) had no effect on oocyte maturation. The unique ability of RIT, among serotonergic antagonists, to induce GVBD did not match the antagonist profile of any known serotonin receptors including Xenopus 5-HT7R, the only known G(s)-coupled serotonin receptor cloned so far in this species. Unexpectedly, injection of x5-HT7R mRNA in frog oocytes resulted in hormone-independent frog oocyte maturation. The addition of exogenous serotonin abolished x5-HT7R-induced oocyte maturation. Furthermore, the combination of x5-HT7R and exogenous serotonin potently inhibited progesterone-induced oocyte maturation. These results provide the first evidence that a G-protein coupled receptor related to 5-HT7R may play a pivotal role in maintaining G2 arrest in vertebrate oocytes.     

The association of serotonin with the alimentary canal of the African migratory locust, Locusta migratoria: distribution, physiology and pharmacological profile

The association of serotonin with the alimentary canal of Locusta migratoria was investigated using immunohistochemistry and high performance liquid chromatography (HPLC) coupled to electrochemical detection. Serotonin-like immunoreactive processes were differentially distributed between and within three regions of the alimentary canal; the foregut, midgut and hindgut. The midgut possessed the most serotonin-like immunoreactive processes, while the hindgut contained only a few immunoreactive processes. Using HPLC coupled to electrochemical detection the serotonin content was highest in the midgut followed by the foregut and hindgut. The physiological response of the midgut to serotonin as well as to the combination of serotonin and proctolin was also examined. It was found that the application of serotonin to the midgut leads to a dose-dependent reduction in tonus of the circular muscles. Serotonin was also able to inhibit a proctolin-induced contraction of the midgut in a dose-dependent manner. The physiological and pharmacological properties of serotonin agonists and antagonists on the midgut were also investigated. The results indicate that alpha-methyl 5-HT was the most effective agonist leading to a 108% relaxation at 10(-9) M compared to that caused by the same serotonin concentration. Among several serotonin receptor antagonists tested, mianserin was the most potent. The application of mianserin at 10(-5) M in combination with 5x10(-6) M serotonin resulted in a 66% reduction of the serotonin-induced relaxation of midgut muscle. The serotonin antagonist cyproheptadine was less effective leading to a 39% reduction of the 5x10(-6) M serotonin-induced relaxation. Ketanserin was a weak antagonist.     

Binding of [3H]Serotonin to Skeletal Muscle Actin

We previously observed that the neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) binds with high- and low-affinity interactions to an actin-like protein prepared from rat brain synaptosomes. In this study, we examined its binding to highly purified actin obtained from rabbit skeletal muscle. Monomeric G-actin bound serotonin with high and low affinities, exhibiting equilibrium dissociation constants (KD values) of 5 X 10(-5) M and 4 X 10(-3) M, respectively. The serotonin binding site on actin was distinct from those sites previously characterized for divalent cations, nucleotides, and cytochalasin alkaloids. The binding of serotonin (1 microM) to G-actin was increased as much as 26-fold by divalent cations. Potassium iodine (KI) increased the affinity of G-actin for serotonin, KD values for this binding being 3 X 10(-7) M and X 10(-5) M. Serotonin bound with even higher affinity to polymerized F-actin, with KD values of 2 X 10(-8) M and 2 X 10(-5) M. However, the total number of binding sites on F-actin was only about 4% of the number of G-actin. The binding of serotonin (0.1 microM) to G-actin could be inhibited by phenothiazines (1 microM) or reserpine (10 microM), but not by classical antagonists of serotonin receptors or by drugs that release serotonin or inhibit its uptake. The binding of serotonin to actin in vivo may participate in a contractile process related to neurotransmitter release.     

Influence of chain length and N-alkylation on the selective serotonin receptor ligand 9-(aminomethyl)-9,10-dihydroanthracene

Comparison of the serotonin 5-HT2A receptor affinities of chain lengthened and N-alkylated analogues of the novel ligand 9-aminomethyl-9,10-dihydroanthracene (AMDA) and a structurally similar prototypical tricyclic amine imipramine suggests that the two agents bind to the receptor in different fashions. The demonstration that AMDA is highly selective for serotonin receptors (5-HT2A, K = 20nM; 5-HT2C, Ki=43nM) versus the dopamine D2 receptor (Ki>10,000nM), as well as the serotonin and norepinephrine transporters (Ki>10,000nM) further suggests that AMDA and the nonselective ligand imipramine interact with these target macromolecules in different ways.     

Characterization of tryptamine 5-hydroxylase and serotonin synthesis in rice plants

Serotonin is a well-known pineal hormone that in mammals plays a key role in mood. In plants, serotonin is implicated in several physiological roles such as flowering, morphogenesis, and adaptation to environmental changes. However, its biosynthetic enzyme in plants has not been characterized. Therefore, we measured the serotonin content and enzyme activity responsible for serotonin biosynthesis in rice seedlings. Tryptamine 5-hydroxylase (T5H), which converts tryptamine into serotonin, was found as a soluble enzyme that had maximal activity in the roots. The maximal activity of T5H was closely associated with the enriched synthesis of serotonin in roots. Tetrahydropterine-dependent T5H activity was inhibited by tyramine, tryptophan, 5-OH-tryptophan, and octopamine, but remained unaltered by dopamine in vitro. The tissues of rice seedlings grown in the presence of tryptamine exhibited a dose-dependent increase in serotonin in parallel with enhanced T5H enzyme activity. However, no significant increase in serotonin was observed in rice tissues grown in the presence of tryptophan, suggesting that tryptamine is a bottleneck intermediate substrate for serotonin synthesis.