Caffeine injection raises brain tryptophan level,but does not stimulate the rate of serotonin synthesis in rat brain

Acute caffeine injection (100 mg/kg) elevates brain levels of tryptophan (TRP), serotonin (5HT), and 5-hydroxyindoleacetic acid (5HIAA). Experiments were performed to determine if the increases in 5HT and 5HIAA result from a stimulation of the rate of 5HT synthesis. Both the rate of 5-hydroxytryptophan (5HTP) accumulation following NSD-1015 injection, and the rate of ³H-5-hydroxyindole synthesis from ³H-tryptophan were measured $\text{in vivo}$ following caffeine administration and found to be normal. Tryptophan hydroxylase activity, as measured $\text{in vitro}$ in brain homogenates, was also unaffected by caffeine. The results suggest that the elevations in brain 5HT and 5HIAA levels produced by caffeine do $\text{not}$ reflect enhanced 5HT synthesis, despite significant elevations in brain TRP level. Some other mechanism(s) must therefore be responsible for these elevations in brain 5-hydroxyindole levels.     

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.     

Tricyclic Imipramine Modification of the Circadian Rhythms of Hypothalamic Serotonin, its Precursors and Acid Catabolite in Individually Housed Rats

Circadian rhythms of serotonin (5HT), its precursors tryptophan (TP) and 5-hydroxy-tryptophan (5HTP) and its acid catabolite 5-hydroxy-indoleacetic acid (5HIAA), were determined in the hypothalamus of control rats and rats which had been treated continuously with subcutaneous imipramine (10 mg/kg/day) for 2 weeks.

Rats were individually housed and entrained to LD12:12. Controls showed the 5HT and TP peaks in the light and dark periods respectively, as reported in the literature, but no inverted correlation (antiphase) between SHT and 5HIAA rhythms.

Imipramine significantly modified circadian rhythm characteristics: the 5HT acrophase was advanced, that of TP and 5HIAA was delayed. Imipramine also significantly increased hypothalamic SHT and TP concentrations.     

Brain Lipid Analysis in Mice with Rett Syndrome

Rett syndrome (RS) is an X-linked neurodevelopmental disorder mostly involving mutations in the gene for methyl-CpG-binding protein 2 (MECP2). Ganglioside abnormalities were previously found in cerebrum and cerebellum in RS patients. We evaluated total lipid distribution in cerebrum/brainstem, hippocampus, and cerebellum in male mice carrying either the Mecp2 ^tm1.1Bird knockout mutation or the Mecp2 ^308/y deletion mutation. The concentration of the neuronal enriched ganglioside GD1a was significantly lower in the cerebrum/brainstem of Mecp2 ^tm1.1Bird mice than in that of age matched controls, but was not reduced in the Mecp2 ^308/y mice. No other differences in brain lipid content, including myelin-enriched cerebrosides, were detected in mice with either type of Mecp2 mutation. These findings indicate that the poor motor performance previously reported in the RS mutant mice is not associated with major brain lipid abnormalities and that most previous brain lipid abnormalities observed in RS patients were not observed in the Mecp2 ^tm1.1Bird or the Mecp2 ^308/y RS mice.     

Cerebrospinal Fluid Concentrations of Tryptophan and 5-Hydroxyindoleacetic Acid in Macaca Mulatta: Diurnal Variations and Response to Chronic Changes in Dietary Protein Intake

In rats, dietary protein is known to influence brain tryptophan (TRP) concentrations and serotonin (5HT) synthesis. However, few studies have examined this relationship in primates (including humans). We therefore studied the effect in monkeys of changes in chronic protein intake on plasma and cerebrospinal fluid (CSF) concentrations of TRP and 5-hydroxyindoleacetic acid (5HIAA), the principal 5HT metabolite. Juvenile male monkeys (Macacca mulatta) consumed for sequential 4-week periods diets differing in protein content (~23% ~ 16% ~10% ~6% protein [%-energy/day]). Each day, food was presented as a morning meal of fruit, and an afternoon meal consisting of a pelleted, commercial diet and fruit. During week 4 on each diet, blood and CSF were sampled diurnally via indwelling catheters. Plasma and CSF TRP varied diurnally and with dietary protein content. On all diets, CSF TRP declined modestly in the morning, and increased in the afternoon; the magnitude of the increments varied directly with dietary protein content. Diurnal variations were absent for CSF 5HIAA; however, CSF 5HIAA varied directly with chronic dietary protein content. We conclude that dietary protein content can chronically influence CSF TRP concentrations in monkeys. The variation in CSF 5HIAA suggests chronic protein intake may influence serotonin synthesis and turnover, perhaps via changes in TRP concentrations.     

Twenty-four hour rhythms in serum and brain indoleamine concentrations: tryptophan-5-hydroxylase and monoamine oxidase activity in the rat.

The relationship between the 24 h rhythm in 5-hydroxy-tryptamine (5HT) levels in rat brain, the availability of precursors of 5HT and the concentration of its major metabolite, 5-hydroxyindole acetic acid (5HIAA) has been investigated. Serum total and "free" tryptophan (TRY) levels and brain TRY levels all show a 24 h rhythm with highest concentrations in the middle of the dark phase i.e. 12 h displaced from that of the 5HT rhythm. No 24 h variation in either tryptophan-5-hydroxylase or monoamine oxidase activity was detected, nor did brain 5-hydroxytryptophan (5HTP) levels vary with clock hour. Changes in 5HIAA concentration paralleled those of 5Ht. The uptake of 14C-5HTP, 14C-TRY and 14C-5HT into homogenates of the septal region of rat brain did not display a circadian rhythm, although there was evidence that uptake of 14C-TRY in an isolated synaptosomal preparation from the same region was greater during the light phase, indicating the possibility that uptake of the precursor into the nerve ending may be, in part, responsible for the 24 h rhythm in brain 5HT. It is concluded that brain 5HT levels are independent of the serum or brain TRY concentrations measured. Since changes in 5HT with clock hour are paralleled by changes in 5HIAA, it also seems unlikely that the increase in brain 5HT during the light phase is caused by a decreased release of 5HT from nerve endings.     

The conversion of [3H] tryptophan to 5-[3H] hydroxytryptamine in mouse brain following depletion of phenylalanine and tyrosine.

Abstract— Phenylalanine ammmonia-lyase (PAL), an enzyme which converts phenylalanine (Phe) and tyrosine (Tyr) to trans-p-cinnamic acid and trans-p-coumaric acid, respectively, was administered to mice and its effect on the conversion of [³H]tryptophan to 5-[³H]HT in the brain was measured. Although PAL significantly depleted plasma Tyr, it has little or no effect on either brain Tyr or catecholamine concentrations. Endogenous brain tryptophan levels were significantly increased 2 h after PAL administration, brain 5-HT was dramatically increased 4 h following PAL and each returned to baseline levels by 8 h. This return to baseline was accompanied by a marked decrease in the fraction of tryptophan converted to 5-HT during a 20 min pulse period preceding death, suggesting the activation of a compensatory decrease in 5-HT synthesis in response to increased 5-HT concentration. These data suggest that PAL administration readily produces reversible alterations in 5-HT synthesis and that this may be a fruitful approach to studying brain 5-HT function.     

The time course of aggressive behaviour in juvenile matrinxã Brycon amazonicus fed with dietary L‐tryptophan supplementation

This study evaluated the influence of dietary L‐tryptophan (TRP) supplementation on the time course of aggressive behaviour and on neuroendocrine and hormonal indicators in juvenile matrinxã Brycon amazonicus. Supplementation with TRP promoted a change in the fight pattern at the beginning of an interaction with an intruder, resulting in decreased aggressive behaviours during the first 20 min. The decrease in aggression did not persist throughout the interaction but increased at 3 and 6 h after the beginning of the fight. Monoamine levels in the hypothalamus were not influenced by TRP before or after the fight; however, the hypothalamic serotonin (5‐HT) concentration and the 5‐hydroxyindole‐3‐acetic acid (5HIAA):5‐HT ratio were significantly correlated with the reduction in aggressive behaviour at the beginning of the fight. Cortisol was not altered by TRP before the fight. After the fight cortisol increased to higher levels in B. amazonicus fed with supplementary TRP. These results indicate that TRP supplementation alters the aggressive behaviour of B. amazonicus and that this effect is limited to the beginning of the fight, suggesting a transient effect of TRP on aggressive behaviour. This is the first study reporting the effects of TRP supplementation on the time course of aggressive interaction in fishes.     

Stereoselective effect of kynurenine enantiomers on the excretion of serotonin and its metabolite in rat urine

A solution of optically pure kynurenine (KYN), i.e., D ‐KYN or L ‐KYN, was administered intravenously to male Sprague‐Dawley rats (10 mg kg^?1 ml^?1). The time‐course of changes in the concentrations of urinary monoamines and their metabolites such as 5‐hydroxytryptamine (5‐HT), 5‐hydroxyindole acetic acid (5‐HIAA), dopamine, and 3‐methoxytyramine were investigated by reversed‐phase high‐performance liquid chromatography with electrochemical detection after precolumn derivatization with (2R)‐2,5‐dioxopyrrolidin‐1‐yl‐2,5,7,8‐tetramethyl‐6‐(tetrahydro‐2H‐pyran‐2‐yloxy)chroman‐2‐carboxylate (NPCA). We observed a stereoselective difference in the effects of the KYN enantiomers. Only D ‐KYN, not L ‐KYN, caused a significant increase in urinary 5‐HT levels within 30 min after its administration. With regard to the metabolites, urinary 3‐MT level was increased by D ‐KYN administration. On the other hand, no significant change in the DA level was observed after administration of either D ‐KYN or L ‐KYN. These results suggest that D ‐KYN could affect the activity of neuroactive amines, especially 5‐HT, in vivo. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Action of tryptophan analogues in Saccharomyces cerevisiae

In an analysis of the effects of various tryptophan and indole analogues in Saccharomyces cerevisiae we determined the mechanisms by which they cause growth inhibition: 4-Methyltryptophan causes a reduction in protein synthesis and a derepression of the tryptophan enzymes despite of the presence of high internal levels of tryptophan. This inhibition can only be observed in a mutant with increased permeability to the analogue. These results are consistent with but do not prove an interference of this analogue with the charging of tryptophan onto tRNA. 5-Methyltryptophan causes false feedback inhibition of anthranilate synthase, the first enzyme of the tryptophan pathway. This inhibits the further synthesis of tryptophan and results in results in tryptophan limitation, growth inhibition and derepression of the enzymes. Derepression eventually allows wild type cells to partially overcome the inhibitory effect of the analogue. 5-Fluoroindole is converted endogenously to 5-fluorotryptophan by tryptophan synthase. Both endogenous and externally supplied 5-fluorotryptophan are incorporated into protein. This leads to intoxication of the cells due to the accumulation of faulty proteins. 5-Fluorotryptophan also causes feedback inhibition of anthranilate synthase and reduces the synthesis of tryptophan which would otherwise compete with the analogues in the charging reaction. Indole acrylic acid inhibits the conversion of indole to tryptophan by tryptophan synthase. This results in a depletion of the tryptophan pool which, in turn, causes growth inhibition and derepression of the tryptophan enzymes.Abbreviations cpm counts per minute - OD optical density at 546 nm - TCA trichloro acetic acid - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for the corresponding tryptophan biosynthetic enzymes - trpl^– res. trp1^± refer to mutant strains synthesizing completely resp. partially defective enzymes     

THE EFFECT OF A LOW PROTEIN DIET AND EXOGENOUS INSULIN ON BRAIN TRYPTOPHAN AND ITS METABOLITES IN THE WEANLING RAT

—Male Wistar rats aged 24 days were divided into three groups. Two groups were given a high protein (250 g/kg casein) and a low protein (30 g/kg casein) diet respectively. The third group was given an amount of the high protein diet containing the same amount of energy as that consumed by the low protein diet rats. The plasma of the animals on low protein contained 20% of the concentration of tryptophan of animals on the other two diets. In these animals the concentration of tryptophan was reduced in the forebrain, cerebellum and brain stem, and the concentrations of 5-HT and 5-hydroxyindoleacetic acid were reduced in the forebrain and brain stem. The low protein diet decreased the total uptake of l -[G-³H]tryptophan into the brain and its incorporation into brain protein. Plasma insulin concentrations were reduced in the low protein and ‘restricted high protein’ animals and the plasma corticosterone concentration was raised in the low protein animals. Exogenous insulin did not raise the plasma tryptophan concentration in the low protein animals but it increased the uptake of l -[G-³H]tryptophan into the brain and its incorporation into protein. Rehabilitation for 7 days restored the plasma and brain tryptophan concentrations and those of brain 5-HT and 5-hydroxyindoleacetic acid to control values.     

Brain serotonin synthesis in MDMA (ecstasy) polydrug users: an alpha‐[11C]methyl‐l‐tryptophan study

3,4‐Methylenedioxymethamphetamine (MDMA, ecstasy) use may have long‐term neurotoxic effects. In this study, positron emission tomography with the tracer alpha‐[¹¹C]methyl‐l ‐tryptophan (¹¹C‐AMT) was used to compare human brain serotonin (5‐HT) synthesis capacity in 17 currently drug‐free MDMA polydrug users with that in 18 healthy matched controls. Gender differences and associations between regional ¹¹C‐AMT trapping and characteristics of MDMA use were also examined. MDMA polydrug users exhibited lower normalized ¹¹C‐AMT trapping in pre‐frontal, orbitofrontal, and parietal regions, relative to controls. These differences were more widespread in males than in females. Increased normalized ¹¹C‐AMT trapping in MDMA users was also observed, mainly in the brainstem and in frontal and temporal areas. Normalized ¹¹C‐AMT trapping in the brainstem and pre‐frontal regions correlated positively and negatively, respectively, with greater lifetime accumulated MDMA use, longer durations of MDMA use, and shorter time elapsed since the last MDMA use. Although the possibility of pre‐existing 5‐HT alterations pre‐disposing people to use MDMA cannot be ruled out, regionally decreased 5‐HT synthesis capacity in the forebrain could be interpreted as neurotoxicity of MDMA on distal (frontal) brain regions. On the other hand, increased 5‐HT synthesis capacity in the raphe and adjacent areas could be due to compensatory mechanisms.

     

Diazepam increases 5-hydroxyindole concentrations in rat brain and spinal cord

Diazepam elevates serotonin (5HT) and 5-hydroxyindoleacetic acid (5HIAA) concentrations in rat brain and spinal cord. The maximal effect occurs 1–2 hrs after drug injection and is dose related between 5–20 mg/kg (intraperitoneal). The action of diazepam on brain 5HT and 5HIAA concentrations is modified by previous food consumption: the ingestion of a diet that raises brain 5HT and 5HIAA one hour before drug injection enhances the diazepam-induced increase in brain indoles; consumption of a diet that lowers brain 5HT and 5HIAA partially blocks the elevation in brain indoles that follows diazepam injection.     

Activation of bulbospinal serotonergic neurons during cold exposure.

In a four-part study, we expand on our previous report that bulbospinal serotonin (5HT) neuronal activation occurs with 24 h of cold exposure. To characterize temporal aspects, rats were exposed to 3 degrees C or were maintained at 22 degrees C for 2, 8, 48, or 96 h (experiment 1) or for 15, 30, or 60 min (experiment 2). To ensure that cold-induced changes in 5HT activity were not due to disturbances in diurnal pattern, rats in experiment 3 were exposed to cold (8 h) during the dark cycle. To explore the hypothesis that cold-induced 5HT activation is part of a broad metabolic response that includes activation of the sympathetic nervous system, metabolically impaired (hypothyroid) rats were exposed to 8 degrees C in experiment 4. Significant increments in 5-hydroxyindoleacetic acid (SHIAA) concentration were evident by 60 min of cold exposure and existed at all later time points measured. These findings were most robust in spinal cord and rostral brainstem. Activation in spinal cord was also found when rats were exposed to 8 h of cold during the dark cycle, the active period for rats. In experiment 4, hypothyroid rats exhibited significantly greater norepinephrine excretion compared with control rats exposed to the same cold stimulus; this finding was accompanied by significantly greater increments in 5HIAA concentration in rostral brainstem and spinal cord of hypothyroid rats. In addition, significant elevations in tryptophan concentration were noted throughout the brainstem and spinal cord of cold-exposed, hypothyroid rats relative to room temperature, hypothyroid rats. This finding suggested that elevations in 5HIAA concentration in these rats were due to increases in precursor availability. The implications of these findings relative to autonomic and metabolic control are discussed.     

Twenty-four-Hour Rhythms of Serum Acth,Prolactin, Growth Hormone,and Thyroid-Stimulating Hormone,and of Median-Eminence Norepinephrine,Dopamine, and Serotonin,in Rats Injected with Freund's Adjuvant

The effect of Freund's adjuvant injection on 24-h variation of circulating ACTH, prolactin, growth hormone (GH), and thyroid-stimulating hormone (TSH) levels, and of norepinephrine (NE) content, and dopamine (DA) and serotonin (5HT) turnover in median eminence, was examined in adult rats kept under light between 0800 and 2000 h daily. Groups of 6–10 animals Freund's complete adjuvant or its vehicle at 1 lOOh 3 days before sacrifice and were killed by decapitation at six different time intervals throughout a 24-h cycle. In rats injected with adjuvant's vehicle, serum ACTH and prolactin exhibited peak values around the light-dark transition (p < 0.0001 and < 0.04, respectively), while the maximum in TSH was found in the late afternoon (p < 0.0001, one-way ANOVA). GH levels did not vary on a 24-h basis. In Freund's adjuvant-injected rats, 24-h variations of TSH levels became blunted, while 24-h variations of prolactin and ACTH persisted. Freund's adjuvant augmented serum ACTH and prolactin levels, and decreased GH and TSH levels (p < 0.0007, factorial ANOVA). Median-eminence NE content, and turnover of DA, assessed by measuring dihydroxyphenylacetic acid, DOPAC/DA ratio, and of 5HT, assessed by measuring 5-hydroxyindoleacetic acid, HIAA/5HT ratio, varied on a 24-h basis in rats receiving adjuvant's vehicle (p < 0.02). Median-eminence NE content attained its maximum at 1600–2000 h, while maxima in DOPA/DA and HIAA/5HT ratios occurred at 0400 h. Injection with Freund's adjuvant reduced the amplitude of the daily variation of NE content, shifted the maximum of DOPAC/DA ratio toward the light-dark transition, and blunted the daily variation in HIAA/5HT ratio in median eminence. The administration at 1200 of the immunosuppressant drug cyclosporine (5 mg/kg, 5 days) restored the augmented ACTH and prolactin levels (p < 0.0001, factorial ANOVA) and depressed GH and TSH levels (p < 0.02) found in Freund's adjuvant-injected rats. Cyclosporine was also effective in restoring 24-h rhythmicity of serum ACTH and TSH, but not of prolactin, levels. Cyclosporine did not modify the effect of Freund's adjuvant on time-of-day changes of median-eminence NE content, but it was effective in counteracting the changes of DA and 5HT turnover found after immunization. The results are compatible with a significant effect of immune-mediated inflammatory response at an early phase after Freund's adjuvant injection on ACTH, GH, prolactin, and TSH release, which is partially sensitive to immunosuppression by cyclosporine. (Chronobiology International, 14(3), 253–265, 1997)     

The late stage following continuous amphetamine administration to rats is correlated with altered dopamine but not serotonin metabolism

In previous experiments rats pretreated with slow-release d-amphetamine (d-Amp) pellets for $\text{4}\text{1}\text{2}$ days, given a 12-hr drug-free period, and then injected with d-Amp have been found to show a behavioral syndrome which has similarities to that induced by acute injections of the hallucinogens LSD and mescaline. The present results indicate that rats administered this same drug regimen have large decreases in Dopamine (DA), dihydroxyphenyl acetic acid (Dopac), and homovanillic acid (HVA) in caudate nucleus, smaller decreases in DA with no changes in Dopac and HVA levels in nucleus accumbens, but no alterations in 5-hydroxytryptamine (5HT) and 5-hydroxyindole acetic acid (5HIAA) levels in caudate, accumbens, brainstem and hippocampus. Increased 5HIAA levels are found in rats sacrificed with pellets intact following 3 days of continuous d-Amp administration, while sleep deprived and in motor stereotypies. The late and hallucinatory stage following continuous d-amp is correlated more closely with alterations in dopamine than of 5HT.     

Heterologous expression of the apple hexose transporter MdHT2.2 altered sugar concentration with increasing cell wall invertase activity in tomato fruit

Sugar transporters are necessary to transfer hexose from cell wall spaces into parenchyma cells to boost hexose accumulation to high concentrations in fruit. Here, we have identified an apple hexose transporter (HTs), MdHT2.2, located in the plasma membrane, which is highly expressed in mature fruit. In a yeast system, the MdHT2.2 protein exhibited high ¹⁴C‐fructose and ¹⁴C‐glucose transport activity. In transgenic tomato heterologously expressing MdHT2.2, the levels of both fructose and glucose increased significantly in mature fruit, with sugar being unloaded via the apoplastic pathway, but the level of sucrose decreased significantly. Analysis of enzyme activity and the expression of genes related to sugar metabolism and transport revealed greatly up‐regulated expression of SlLIN5, a key gene encoding cell wall invertase (CWINV), as well as increased CWINV activity in tomatoes transformed with MdHT2.2. Moreover, the levels of fructose, glucose and sucrose recovered nearly to those of the wild type in the sllin5‐edited mutant of the MdHT2.2‐expressing lines. However, the overexpression of MdHT2.2 decreased hexose levels and increased sucrose levels in mature leaves and young fruit, suggesting that the response pathway for the apoplastic hexose signal differs among tomato tissues. The present study identifies a new HTs in apple that is able to take up fructose and glucose into cells and confirms that the apoplastic hexose levels regulated by HT controls CWINV activity to alter carbohydrate partitioning and sugar content.     

Melatonin synthesis in the optic lobes and midbrain of the grasshopper Oedipoda caerulescens

The pathways of insect melatonin (MEL) biosynthesis apparently follow the same routes as those identified in vertebrates but information on MEL synthesis variations related with serotonin (5‐HT), 5‐hydroxy‐indole acetic acid (5HIAA), and N‐acetylserotonin (NAS) levels, as well as 5‐HT N‐acetyltransferase (NAT) activity throughout the day, is very limited in the insect nervous system. In the present study, the levels of MEL, metabolites (5‐HT, NAS, and 5‐HIAA) and enzyme NAT were determined in the optic lobes and the midbrain of the grasshopper Oedipoda caerulescens, in conditions of light and darkness. In both tissues, a different pattern of MEL synthesis was observed over the light/dark cycle. Variations in the levels of 5‐HT, NAS and NAT activity related to the synthesis of cerebral MEL follow a pattern very similar to that observed in the pineal of mammals, with a peak of synthesis in the first half of the scotophase. Also, we observed differences in the metabolism of 5‐HT between the optic lobes and the midbrain light/dark‐dependent.