Urinary excretion patterns of 5-hydroxyindole-3-acetic acid and 5-hydroxytryptophol in various animal species: implications for studies on serotonin metabolism and turnover rate

The concentrations of the serotonin metabolites 5-hydroxyindole-3-acetic acid (5HIAA) and 5-hydroxytryptophol (5HTOL) were determined in spot urine samples of 12 mammalian and one fish species (cat, cow, dog, ferret, golden hamster, guinea pig, horse, monkey, mouse, rabbit, rainbow trout, rat, sheep) and compared with human data. The highest urinary concentrations of 5HTOL were found in the Sprague-Dawley rat (mean 9.5 micromol/L) and NMRI mouse (8.2 micromol/L), and the lowest in rainbow trout, cynomolgus macaque, and human urine (approximately 0.1 micromol/L). The highest 5HIAA concentrations were found in hamster (89.3 micromol/L) and mouse (85.2 micromol/L), and the lowest in rainbow trout, horse and sheep (range 2.0-3.7 micromol/L). Several species showed 5HIAA concentrations similar to that normally observed in human urine (approximately 5-40 micromol/L). This study demonstrated wide inter- and intra-species variations in the urinary concentrations of 5HIAA and 5HTOL, both separately and in the sum of concentrations. The 5HTOL/5HIAA ratio, which is used as an easily accessible index of the relative importance of the reductive and oxidative pathways for serotonin metabolism, also varied considerably between different species. This observation confirms that the much higher urinary 5HTOL/5HIAA ratio in rats (mean 0.35) compared with humans (< 0.01) is due to a higher baseline formation of 5HTOL in the rat. The monkey, ferret, hamster, and rabbit most closely resembled humans in this respect, and at least the two latter species appear to be more suitable than rats as animal models for studying serotonin metabolism and turnover rate, and the metabolic interaction with ethanol.     

Effects of Chronic Restraint Stress on Feeding Behavior and on Monoamine Levels in Different Brain Structures in Rats

Monoaminergic systems are important modulators of the responses to stress. Stress may influence feeding behavior, and the involvement of monoamines in the control of food intake is well recognized. We investigated the effects induced by chronic-restraint stress, 1 h a day, for 40 days, on eating behavior and on monoamines in distinct brain structures. Increased consumption of sweet pellets, and not of peanuts, was observed. Dopamine (DA), serotonin (5–HT), and their metabolites were measured by HPLC-EC. After chronic restraint, the results observed were decreased 5–HT in hippocampus, with increased 5–HIAA/5–HT; decreased 5–HIAA levels in cortex; reduction in DA in hippocampus, and increased levels in amygdala and hypothalamus; HVA increased in cortex, as well as HVA/DA ratio, while DOPAC/DA decreased. HVA decreased in hypothalamus, as well as HVA/DA, and DOPAC/DA and HVA/DA decreased in the amygdala. These results suggest that restraint stress differentially affects the activity of central dopaminergic and serotonergic neurons, and this may be related to the effects observed in eating behavior.     

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)     

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.     

Biological Actions of Drugs Affecting Serotonin and Eating

The present status of knowledge on drugs affecting food intake and presumably acting via a serotoninergic mechanism is reviewed. The mechanism of action of these drugs is analyzed at the neurochemical level. All the drugs, to various extents, inhibit the uptake of serotonin (5HT), increase the release of 5HT and decrease brain levels of 5HT and 5HIAA. However, the underlying mechanisms are not identical as exemplified by comparisons made with d-fenfluramine, d-norfenfluramine, fluoxetine, sertraline and paroxetine. An analysis of the role of 5HT in the inhibition of food intake reveals that only d-fenfluramine is inhibited by antiserotonin agents. The role of the different 5HT receptor-subtypes in this antagonism is discussed. More selective 5HT antagonists are needed to establish which 5HT receptor(s) controls food intake.     

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.     

Simultaneous determination of norepinephrine,serotonin, and 5-hydroxyindole-3-acetic acid in microdialysis samples from rat brain by microbore column liquid chromatography with fluorescence detection following derivatization with benzylamine

A microbore column liquid chromatographic method for the simultaneous determination of norepinephrine (NE), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5HIAA) in microdialysis samples from rat brain is described. The method is based on precolumn derivatization of NE, 5HT, and 5HIAA with benzylamine in the presence of potassium hexacyanoferrate(III) resulting in the corresponding highly fluorescent and stable benzoxazole derivatives. A 15-microl sample was mixed with 15 microl derivatization reagent solution containing 0.3M 3-cyclohexylaminopropanesulfonic acid buffer (pH 12.0), 0.5M benzylamine, 10mM potassium hexacyanoferrate(III), and methanol (1/1/1/12, v/v/v/v). The derivatization was carried out at 50 degrees C for 20 min. The benzylamine derivatives of NE, 5HT, and 5HIAA were separated on a reversed-phase column (100 x 1.0mm i.d., packed with C18 silica, 5 microm) within 30 min. The mobile phase consisted of 15 mM acetate buffer (pH 5.0) and acetonitrile (31%, v/v); the flow rate was 50 microl/min. The detection limits (signal-to-noise ratio of 3) for NE, 5HT, and 5HIAA in the injection volume of 20 microl were 90, 210, and 260 amol, respectively. Microdialysis samples were collected in 7.5-min intervals from the probes implanted in the hippocampus and prefrontal cortex of awake rats. The basal levels of NE, 5HT, and 5HIAA in the dialysates from the hippocampus were 4.2+/-0.5, 4.9+/-0.6, and 934.1 +/- 63.4 fmol/20 microl, and those from the prefrontal cortex were 6.0+/-1.2,5.51.3, and 669.1 +/- 96.0 fmol/20 microl (mean +/- SE, n=25), respectively. The NE and 5HT levels were altered by perfusion of high-potassium or low-calcium solution and following antidepressant drugs imipramine and desipramine. It is concluded that the new fluorescence derivatization method in combination with microbore column liquid chromatography allows the simultaneous determination of NE, 5HT, and 5HIAA in the microdialysis samples at higher sensitivity, providing easier maintenance in routine use than that achieved by high-performance liquid chromatographic methods with electrochemical detection.     

Serotonin in cisternal cerebrospinal fluid of the rat: measurement and use as an index of functionally active serotonin

A simple, selective reverse-phase HPLC-fluorometric method is described for the determination of serotonin (5HT) in cisternal CSF of the rat. The mean (+/- SE) value of CSF 5HT observed in control adult rats was 457 +/- 83 pg/ml (N = 16). In an attempt to validate the measure as an index of extracellular, or functionally active, 5HT, groups of animals were treated with fenfluramine, amitriptyline, pargyline, pargyline plus tryptophan, and 5-hydroxytryptophan plus carbidopa. In all cases CSF 5HT appeared to reflect well the presumed effects of the agents on extracellular levels of 5HT. CSF 5HT was superior in this regard to brain 5HT, brain 5HIAA, or CSF 5HIAA levels. The measurement of cisternal CSF 5HT would appear to offer a convenient index of functionally active 5HT.     

Role of some biogenic amines in the pathomechanism of glomerulonephritis. II. Adrenaline, noradrenaline and serotonin in acute serum sickness in rabbits

In acute serum sickness induced with one intravenous dose of bovine serum albumin (BSA) in 40 rabbits the patterns of excretion of adrenaline (A), noradrenaline (NA), serotonin (5HT) and 5-hydroxyindole-acetic acid (5HIAA) were studied in 24-hour urine, and the 5HT level was determined at intervals of three days. The urinary levels of biogenic amines were determined daily. Some rabbits immunized with BSA received also additionally 5HT, reserpine or parachlorphenylalanine (PCPA). Administration of BSA to rabbits caused a significant increase in the excretion of A and NA and a less evident increase in 5HT level in the blood. The greatest correlation with the course of the immune reaction was shown by the increase in NA excretion observed on the 2nd and 3rd days after BSA administration, and then between the 5th and the 12th days of the experiment. Daily subcutaneous injections of 5HT during 15 days caused a significant rise of its level in the blood and urine, and an increase of 5HIAA excretion. After reserpine or PCPA administration a significant decrease of the levels of all these amines was observed. Taking into account the results of histological examination of the kidneys, that is intensification of the inflammatory changes after 5HT administration and evident inhibition of the inflammatory process after administration of reserpine and PCPA it must be accepted that the studied amines have an important role in the pathomechanism of glomerulonephritis in acute serum sickness.     

High doses of alcohol increase urinary testosterone-to-epitestosterone ratio in females

The effect of alcohol (1.2 and 2.0 g/kg) on the urinary testosterone-to-epitestosterone (T/E) ratio was studied by two experiments each conducted with four healthy females and males. The intake of 2.0 g/kg of ethanol within 5 h in the evening significantly increased plasma testosterone concentration and ratio of T/E in urine collected next morning in females. The results suggest that alcohol increases the T/E ratio more in females than in males. The effect of high doses of alcohol on urinary T/E ratio must be kept in mind when doping tests are performed during training periods.     

Regional Variations of 5HT Concentrations in Rora sg (staggerer) Mutants

Ataxic Rora ^sg (staggerer) mouse mutants, containing a deletion of the Rora gene which encodes a retinoid-like nuclear receptor, were compared to non-ataxic controls for concentrations of 5-hydroxytryptamine (HT), its main metabolite (5-hydroxy-indole acetic acid, 5HIAA), and its precursor (tryptophan) in cerebellum, brainstem, and forebrain. In Rora ^sg cerebellum, 5HT concentrations increased relative to controls, while tryptophan concentrations decreased. 5HIAA concentrations increased in mutant cerebellum and brainstem, but the 5HIAA/5HT ratio declined only in cerebellum. These results indicate that 5HT turnover decreased in cerebellum of an ataxic mutant, perhaps indicative of presynaptic accumulation and compromised neurotransmission and susceptible to be modified by 5HT pharmacotherapy.     

Chronic morphine administration decreases 5-hydroxytryptamine and 2-hydroxyindoleacetic acid content in the brain of rats

To study the effects of chronic morphine treatment on cerebral 5-hydroxytryptamine (5HT) metabolism morphine was administered twice daily for 5 or 8 weeks to male Wistar rats. Control rats were treated with 0.9% NaCl solution for the same period. In rats treated chronically with morphine for 8 weeks the cerebral concentrations of 5HT and 5HIAA were reduced by 12--15% (P less than 0.05) at 26--28 h after the last morphine injection (50 mg/kg s.c.). No such decrease was found in the brain of rats treated with morphine for 5 weeks. A test dose of morphine (30 mg/kg s.c. 2h) increased the cerebral concentration and probenecid-induced accumulation of 5HIAA in the rats treated with morphine for 8 weeks almost as much as in the brain of the control rats. Naloxone (10 mg/kg s.c. 2h) did not cause clear changes in the cerebral 5HT or 5HIAA concentration. These experiments suggest that endogenous opioid mechanisms are concerned in the regulation of 5HT neurons and that prolonged morphine treatment weakens these mechanisms. This weakening of endogenous regulation of 5HT neurons, which, however, still respond to acute morphine administration, might be part of the mechanism of compulsive drug use in narcotic addiction. It is possible that these neurons in dependent individuals do not function optimally without exogenous morphine. A similar phenomenon--weakening of endogenous regulation combined with clear responsivity to exogenous opiates--occurs in the cerebral dopamine neurons of rats treated chronically with narcotic analgesics.     

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.     

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.     

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.     

Correlations of serotonin and its metabolites in individual rat pineal glands over light:dark cycles and after acute light exposure

Profiles of pineal indolealkylamines were estimated by high performance liquid chromatography and were correlated in individual glands of male rats sacrificed over several light:dark cycles and after acute exposure to light at night. A significant and positive correlation of 5HIAA vs 5HT in individual glands over both normal and experimental lighting conditions suggested that oxidative deamination is not a major factor in photic regulation of pineal 5HT levels and that the formation of 5HIAA is dependent on substrate availability. Regression analysis of other indole constituents revealed that there was a positive and significant correlation between 5HT vs N-acetylserotonin, but not between 5HT vs melatonin and N-acetylserotonin vs melatonin in individual glands during the dark phase of a light:dark cycle. We propose that this effect may be related to a pulsatile release of melatonin into the blood stream and is the result of sampling glands at different stages in the storage/release of melatonin.     

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.     

Effects of acute handling stress on cerebral monoaminergic neurotransmitters in juvenile Senegalese sole Solea senegalensis

Juvenile Senegalese sole Solea senegalensis were subjected for short periods to two different types of handling‐related stress: air exposure stress and net handling stress. The S. senegalensis were sacrificed 2 and 24 h after the stress events and the levels of serotonin (5‐HT), noradrenaline (NA), dopamine (DA) and their respective major metabolites, 5‐hydroxyindoleacetic acid (5‐HIAA), 3‐methoxy‐4‐hydroxyphenylglycol (MHPG) and 3,4‐dihydroxyphenylacetic acid (DOPAC), were measured in three brain regions (telencephalon, hypothalamus and optic tectum) and compared with those in control, non‐stressed S. senegalensis. Neither type of stress caused any significant alteration of serotoninergic activity (5‐HIAA:5‐HT ratio) or NA levels. Dopaminergic activity (DOPAC:DA ratio) was lower in stressed fish in all of the brain regions studied. For both air exposure stress and net handling stress, DA levels were significantly higher (P < 0·05) than in the control S. senegalensis. In addition, the higher DA levels after net handling stress were always significantly higher (P < 0·05) than those observed after acute air exposure stress, except in the telencephalon after 24 h. The significantly lower DOPAC:DA ratio (P < 0·05) in all of the brain regions studied was only observed in response to net handling stress.     

The influence of sex and spawning on levels of tryptophan, serotonin and 5-hydroxyindoleacetic acid in the brains of wild brook trout, Salvelinus fontinalis

Levels of tryptophan (TP), serotonin (5-hydroxytryptamine, 5HT) and 5-hydroxyindoleacetic acid (5HIAA) have been determined in the brains of wild brook trout, Salvelinus fontinalis (Mitchill), and brown trout, Salmo trutta L., using high performance liquid chromatography with electrochemical detection. Immediately prior to spawning, adult female brook trout exhibit higher levels of 5HT in the brain than adult males, immature brook trout and immature brown trout. After spawning, the highest levels of TP are found in spent males, which also have higher levels of 5HT in the brain than spent females and immature brook trout. Immature brook trout exhibit higher levels of 5HIAA than prespawning adults. This difference disappears after the spawning season. Serum protein levels and condition factors are lower in spent female brook trout; however, haematocrit values for both sexes remain unchanged after spawning.     

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.