Monoamines and Their Precursors and Metabolites in the Chicken Brain, Pineal, and Retina: Regional Distribution and Day/Night Variations

Levels of norepinephrine, epinephrine, dopamine, and serotonin (5-HT) and their precursors [tyrosine, L-3,4-dihydroxyphenylalanine, tryptophan, and 5-hydroxytryptophan (5-HTP)] and metabolites [3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), homovanillic acid, 3-methoxy-4-hydroxyphenylglycol, and 5-hydroxyindoleacetic acid (5-HIAA)] were determined concurrently in samples of chick retina, pineal gland, and nine selected areas of the brain (optic lobes, thalamus, hypothalamus, optic chiasm, pons/medulla, cerebellum, neostriatum/ectostriatum, hyperstriatum, and basal forebrain) using HPLC coupled with a coulometric electrode array detection system. The norepinephrine level was highest in the pineal gland, but it was also widely distributed throughout the chick brain, with the thalamus and hypothalamus showing substantial levels. The dopamine level was highest in the basal forebrain. The epinephrine level was highest in the hypothalamus. The thalamus and hypothalamus showed the highest levels of 5-HT. Daytime levels (1100 h) of these compounds were compared with levels in chicks killed in the middle of the dark phase (2300 h). In the brain areas examined, no day/night variations in levels of norepinephrine, epinephrine, dopamine, or 5-HT were seen, although significant nocturnal changes in levels of their metabolites were observed in some areas. Pineal levels of 5-HIAA decreased significantly at night. The retina showed significant nocturnal increases in 5-HTP, 5-HT, and 5-HIAA levels. Retinal levels of 3-MT and DOPAC were significantly decreased at night.     

Seasonal rhythms of the indolamine content in the epiphysis of rats

Pineal function was studied in Wistar pubertal male rats in summer and winter. It was demonstrated that the pineal weight is inversely related to the day length, increasing in winter and reducing in summer. In winter pineal serotonin is actively metabolized via N-acetylation and subsequent ortho-methylation to form N-acetyl serotonin and melatonin as well as via oxidative deamination followed by ortho-methylation to form 5-hydroxyindolacetic and 5-methoxyindolacetic acids. In summer serotonin metabolism in the pinealocytes changes to direct ortho-methylation of serotonin to form 5-methoxytryptamine. The decreased level of N-acetyltransferase limits the formation of melatonin in the pineal gland in summer.     

Effect of lithium oxybutyrate on the serotonin and 5-hydroxyindoleacetic acid content in the brain of rabbits

Single administration of lithium hydroxybutyrate (10 mg/kg) to rabbits decreased serotonin and 5-hydroxyindoleacetic acid (5-HIAA) content in the caudate nucleus. The drug administration for 8 days is accompanied by mediator accumulation in the cortex, caudate nucleus, tonsils, hypothalamus, thalamus, and midbrain with parallel reduction in 5-HIAA level in these structures. 15 days of lithium hydroxybutyrate administration lead to the increase of serotonin and 5-HIAA concentration, while 28 days of administration reduced the content of mediator and its metabolite.     

Effects of transient, global, cerebral ischemia on striatal extracellular dopamine, serotonin and their metabolites

Rat striatal extracellular fluid levels of dopamine, serotonin, 3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were measured before, during and after transient, global cerebral ischemia in awake rats using in vivo brain microdialysis. Before ischemia, extracellular levels of dopamine, DOPAC, HVA and 5-HIAA were detectable and consistent from sample to sample. During cerebral ischemia, there was a large increase in extracellular dopamine levels and a decrease in the extracellular levels of DOPAC, HVA, and 5-HIAA. During reperfusion, dopamine levels returned to normal as did those of DOPAC, HVA and 5-HIAA. Dialysate serotonin and 3-methoxytyramine concentrations were below detection limits except for samples collected during ischemia and early reperfusion.     

The organochlorine insecticide 1,2,3,4,5,6-hexachlorocyclohexane (lindane) but not 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) augments the nocturnal increase in pineal N-acetyltransferase activity and pineal and serum melatonin levels

The effect of organochlorine insecticides lindane (1,2,3,4,5,6-hexachlorocyclohexane) and DDT (1,1,1-trichloro-2,2-bis (p-chlorophenyl)ethane) were studied in terms of their effects on the rat pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity and pineal and serum melatonin levels during the day (2000h) and at night (2300 and 0100h). Additionally, pineal levels of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), and 5-hydroxyindole acetic acid (5-HIAA) were estimated. Nocturnal NAT activity was increased after lindane administration; likewise, lindane augmented pineal and serum melatonin levels at 2300h. Conversely, DDT was without a statistically significant effect on either NAT activity or on pineal or serum melatonin levels. Neither lindane nor DDT significantly influenced pineal HIOMT values either during the day or at night. Likewise, neither insecticide consistently influenced pineal levels of either 5-HTP, 5-HT or 5-HIAA. The results indicate that the organochlorine insecticide, lindane, modifies pineal melatonin synthesis in vivo.     

Effects of acute ethanol administration on nocturnal pineal serotonin N-acetyltransferase activity

The effect of acute ethanol administration on pineal serotonin N-acetyltransferase (NAT) activity, norepinephrine and indoleamine content was examined in male rats. When ethanol was administered in two equal doses (2 g/kg body weight) over a 4 hour period during the light phase, the nocturnal rise in NAT activity was delayed by seven hours. The nocturnal pineal norepinephrine content was not altered by ethanol except for a delay in the reduction of NE with the onset of the following light phase. Although ethanol treatment led to a significant reduction in nocturnal levels of pineal serotonin content, there was no significant effect upon pineal content of 5-hydroxyindoleacetic acid (5-HIAA). The data indicate that ethanol delays the onset of the rise of nocturnal pineal NAT activity.     

Urinary excretion of bioactive amines and their metabolites in psychiatric patients receiving phenelzine

Phenelzine [2-phenylethylhydrazine] (PLZ), a potent inhibitor of monoamine oxidase (MAO)-A and-B, is used widely in psychiatry. We have studied the effects of PLZ administration on urinary excretion of several bioactive amines and their metabolites in psychiatric patients. Urine samples (24-hour) were collected prior to treatment and again at 2 and 4 weeks of treatment with PLZ (30–90 mg daily in divided doses). Amines and metabolites analyzed included 2-phenylethylamine (PEA), m-and p-tyramine (m-and p-TA), phenylacetic acid (PAA), m-and p-hydroxyphenylacetic acid (m-and p-OH-PAA), tryptamine (T), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), normetanephrine (NME), 3-methoxy-4-hydroxyphenylglycol (MHPG), 3-methoxytyramine (3-MT), and homovanillic acid (HVA). Levels of PEA, p-TA, 5-HT, and T were elevated during treatment with PLZ, but no significant changes in urinary excretion of the acid metabolites PAA, p-OH-PAA, and 5-HIAA were observed. Urinary levels of the noradrenaline metabolites NME and MHPG were increased and decreased, respectively; a similar pattern was observed with the dopamine metabolites 3-MT and HVA. There was an elevation in levels of m-TA and a decrease in its acid metabolite m-OH-PAA during the treatment with PLZ.     

Epiphysis cerebri-thyroid interrelations. Distorting effect of partial thyroidectomy on the antithyroid action of epiphyseal methoxyindoles in animal experiments

The effect of pineal methoxyindoles (MI) on hypothalamus-hypophysis-thyroid gland system was studied in intact and partially thyroidectomized male Wistar rats in conditions of a short light day (winter). Melatonin administration for 10 days suppressed 131I uptake by the thyroid gland and decreased the levels of T3, T4 and thyrotropic hormone (TTH) in the blood serum of intact animals, with TTH reaction to thyroid hormone (TH) retained. 5-methoxytryptamine administration was less effective. Partial thyroidectomy distorted the direction of MI effect: melatonin and to a lesser extent 5-methoxytryptamine caused a marked normalization of a decreased TH content and an increased TTH level in the blood serum of partially thyroidectomized rats. TTH-TH reaction also corresponded to the control. A predominantly modulating character or pineal MI effect on thyroid system is suggested.     

Formation and Clearance of Interstitial Metabolites of Dopamine and Serotonin in the Rat Striatum: An In Vivo Microdialysis Study

In vivo microdialysis was employed in order to characterize the steady-state kinetics of the turnover of specific dopamine and serotonin metabolites in the rat striatum 48 h after surgery. Inhibitors of monoamine oxidase (MAO; pargyline) and catechol-O-methyltransferase (COMT; Ro 40-7592) were administered, either separately or in conjunction, at doses sufficient to block these enzymes in the CNS. In some experiments, the acid metabolite carrier was blocked with probenecid. Temporal changes were then observed in the efflux of interstitial dopamine, 3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA). The fractional rate constants for the accumulation or disappearance of the metabolites could be determined after pharmacological blockade of catabolic enzymes or the acid metabolite carrier. Interstitial 5-HIAA was found to be cleared with a half-life of approximately 2 h. After blockade of either MAO or COMT, HVA disappeared with a half-life of 17 min. Experiments employing probenecid suggested that some of the interstitial HVA was cleared by the acid metabolite carrier, the remainder being cleared by a probenecid-insensitive process, possibly conjugation. After MAO inhibition, DOPAC disappeared with an apparent half-life of 11.3 min. The rate of 3-MT accumulation after pargyline indicated that the majority of interstitial HVA (> 95%) is formed from DOPAC rather than 3-MT. The formation of 3-MT from interstitial dopamine, calculated from the accumulation rate of 3-MT after pargyline, appeared to follow first-order kinetics (k = 0.1 min-1).     

Production of 5-hydroxyindoleacetic acid from serotonin by cultured endothelial cells

Endothelial cells cells from bovine aorta and human umbilical vein and fibroblasts from human foreskin were cultured and subsequently evaluated for ability to metabolize serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA). Cells were incubated for three hours with 4 X 10(-6) M [14C] 5-HT creatinine sulfate. [14C] 5-HIAA was separated from labeled 5-HT by column chromatography and measured for scintillation counting. Production of 5-HIAA by bovine aorta cells was 39.0+/-7.5 (S.E.M., n=6) nmoles per 10(9) cells per hour. Production of 5-HIAA was markedly inhibited by the presence of 10(-4) M iproniazid (an inhibitor of monoamine oxidase) or 10(-4) M imipramine (an inhibitor of amine transport). 5-HIAA was the only product of 5-HT metabolism detected by thin layer chromatography. Production of 5-HIAA by human umbilical vein endothelial cells was 5.4+/-2.0 nmoles per 10(9) cells per hour (n=5) and by human foreskin fibroblasts was 3.9+/-1.4 nmoles per 10(9) cells per hour (n=5). The results obtained during incubation in the presence and absence of inhibitors indicate that bovine aorta endothelial cells maintained in tissue culture are able to transport serotonin with subsequent production of 5-HIAA. By contrast, human umbilical vein endothelial cells and fibroblasts exhibited relatively low rates of 5-HT uptake and metabolism.     

Polyunsaturated fatty acids (PUFA) regulate neurotransmitter contents in rat brain

The effects of feeding of 6-propylthiouracil (6-PTU) and polyunsaturated fatty acids (PUFA) independently and in combination and administration (ip) of a single dose of triiodothyronine (T3) (2.5 microg/100 g body wt) along with feeding of 6-PTU and PUFA were studied in rat brain. Dopamine (DA), 5-hydroxytryptophan (5-HTP), serotonin (5-HT), 5-hydroxy indole acetic acid (5-HIAA), norepinephrine (NE) and epinephrine (EPI) contents were assayed in the hypothalamus and cerebral cortex regions. It was found that 6-PTU feeding resulted in decrease in dopamine, 5-HT, 5-HTP and 5-HIAA in both regions. In animals fed with PUFA followed by administration of T3, the DA level was found normal.     

Neuroendocrine integrative mechanisms in mammalian pineal gland: effects of steroid and adenohypophysial hormones on melatonin synthesis in vitro

The time course for the decrease in norepinephrine concentration of rat pineal explants in culture indicated a significant fall starting at the 4th hour and completed after 16-24 h of incubation. Significant decreases of serotonin and 5-hydroxyindoleacetic acid (HIAA) levels in tissue, an increase of HIAA/serotonin ratio, and an increase of melatonin production rate in vitro were also observed as a function of the incubation time. Estradiol (10(-7)-10(-5) M) increased rat pineal melatonin content, testosterone (10(-5) M) decreased it and progesterone was devoid of activity when incubated with explants for up to 6 h. The in vitro stimulatory effect of estradiol on rat pineal methoxyindole synthesis was blocked by propranolol but not by phentolamine; propranolol also blocked the increase of nuclear estradiol-receptor complex produced by estrogen exposure of pineal explants. TSH (1-100 ng/ml), growth hormone (10-100 ng/ml) and LH (10 ng/ml) augmented rat pineal melatonin content while 100 ng/ml of FSH decreased it significantly. Prolactin exerted a biphasic effect on rat pineal explants, the lowest concentration augmenting melatonin content while the high concentration depressed it. Deep, intermediate and superficial segments of guinea-pig pineal glands showed an increase in melatonin concentration after a 6-h incubation in the presence of 10(-7)-10(-5) M estradiol.     

Effects of an N-methyl-d-aspartate receptor agonist and its antagonist CPP on the levels of dopamine and serotonin metabolites in rat striatum collected in vivo by using a brain dialysis technique

3-((±)-2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) is an antagonist at the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. In the present study, levels of dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA) were measured after intracerebroventricular injection of NMDA, CPP or both in rat striatum using a brain dialysis method. The injection of NMDA produced a significant increase in DOPAC level. HVA level was also increased by NMDA injection. The level of 5-HIAA was not affected by NMDA injection. The injection of CPP had no effect on DOPAC, HVA and 5-HIAA levels. The injection of CPP restrained the increase of DOPAC and HVA levels induced by NMDA injection. The results suggest that intracerebral injection of NMDA may increase dopamine release from rat striatum, but have no effect on serotonin release. Furthermore, CPP inhibits NMDA induced release of dopamine.     

Biogenic monoamine levels in the central nervous system of the sea hare,Aplysia kurodai

1. By using a three-dimensional-coulometric HPLC system, biogenic monoamines and their metabolites were quantified simultaneously in the central nervous system of the sea hare, Aplysia kurodai.2. Precursor amino acids, tyrosine-4 (TYR-4) and tryptophan (TRP), and dopamine (DA), 3, 4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxytryptamine (5-HT) were detected in all the ganglia examined.3. Levels of these compounds in the cerebral, pedal and parieto-visceral ganglia were higher than those of the other ganglia examined.4. In some ganglia, epinephrine (E), 3-O-methyldopa (30MD), 3-methoxytyramine (3-MT), dihydroxyphenylethleneglycol (DOPEG), metanephrine (MN), vanillic acid (VA), octopamine (OCT), kynurenine (KYN) and 5-hydroxyindoleacetic acid (5-HIAA) were also detected.5. The main metabolic pathways of biogenic monoamines were shown to be TYR-4DADOPAC and TRP5-HT5-HIAA. Furthermore, following five pathways were also suggested to be present; TYR-4DAEMNVA, TYR-4TYRAOCT, TYR-43OMD, DA3-MT. EDOPEG and TRPKYN.     

The Effect of Curcumin on Ethanol Induced Changes in Suprachiasmatic Nucleus (SCN) and Pineal

(1) Circadian clocks have been localized to discrete sites within the nervous system of several organisms and in mammals to the suprachiasmatic nucleus (SCN) in the anterior hypothalamus. The SCN controls and regulates the production and discharge of melatonin (hormonal message of darkness) from the pineal gland via a multisynaptic efferent pathway. The nocturnal rise in melatonin production from serotonin results due to an increased activity of serotonin N-acetyl transferase (NAT). (2) The complex interaction between alcohol and biological clock need to be understood as alcoholism results in various clock linked neuronal disorders especially loss of memory and amnesia like state of consciousness, sleep disorders, insomnia, dementia etc. (3) Serotonin, 5-Hydroxy-tryptamine (5-HT) plays an important role in mediating alcohol’s effects on the brain. Understanding the impact of alcohol consumption on circadian system is a pre-requisite to help in treatment of alcohol induced neurological disorders. We, therefore, studied the effect of ethanol drinking and ethanol withdrawal on daily rhythms of serotonin and its metabolite, 5-hydroxy-indole acetic acid (5-HIAA) in SCN and Pineal of adult male Wistar rats maintained under light-dark (LD, 12:12) conditions. (4) Curcumin is well known for its protective properties such as antioxidant, anti-carcinogenic, anti-viral and anti-infectious etc. Hence, we studied the effect of curcumin on ethanol induced changes on 5-HT and 5-HIAA levels and rhythms in SCN and Pineal. (5) Ethanol withdrawal could not restore either rhythmicity or phases or levels of 5-HT and 5-HIAA. Curcumin administration resulted in partial restoration of daily 5-HT/5-HIAA ratio, with phase shifts in SCN and in Pineal. Understanding the impact of alcohol consumption on circadian system and the role of herbal medication on alcohol withdrawal will help in treatment of alcohol induced neurological disorders.     

In vivo release of endogenous dopamine, 5-hydroxytryptamine and some of their metabolites from rat caudate nucleus by phenylethylamine

The in vivo release of endogenous 3,4-dihydroxyphenylethylamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 3-methoxytyramine (3-MT), and of 5-hydroxytryptamine (5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), has been measured in the caudate nucleus of the anesthetized rat. A push-pull cannula was implanted into the brain, and the tissue perfused with artificial CSF or artificial CSF containing 5×10^–4 M phenylethylamine. The perfusate was collected and analyzed for DA, 5-HT and their metabolites by high performance liquid chromatography with electrochemical detection (HPLC-ECD). DA was released by phenylethylamine at rates significantly greater than its basal rate. 3-MT and 5-HT were undetectable in perfusates collected under basal conditions, but could be detected readlly during phenylethylamine stimulation. DOPAC, HVA and 5-HIAA concentrations were not significantly affected by phenylethylamine. The results suggest (1) that phenylethylamine may exert its behavioural effects through increased release of both DA and 5-HT, and (2) that in vivo measurements of the acid metabolites alone may not be indicative of the release of the amines.Special Issue Dedicated to Dr. Abel Lajtha.     

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.     

Photoperiod affects amplitude but not duration of in vitro melatonin production in the ruin lizard (Podarcis sicula)

The pineal gland and its major output signal melatonin have been demonstrated to play a central role in the seasonal organization of the ruin lizard Podarcis sicula. Seasonal variations in the amplitude of the nocturnal melatonin signal, with high values in spring as compared to low values in summer and autumn, have been found in vivo. The authors examined whether the pineal gland of the ruin lizard contains autonomous circadian oscillators controlling melatonin synthesis and whether previously described seasonal variations of in vivo melatonin production can also be found in isolated cultured pineal glands obtained from ruin lizards in summer and winter. In vitro melatonin release from isolated pineal glands of the ruin lizard persisted for 4 days in constant conditions. Cultured explanted pineal glands obtained from animals in winter and summer showed similar circadian rhythms of melatonin release, characterized by damping of the amplitude of the melatonin rhythm. Although different photoperiodic conditions were imposed on ruin lizards before explantation of pineal glands, the authors did not find any indication for corresponding differences in the duration of elevated melatonin in vitro. Differences were found in the amplitude of in vitro melatonin production in light/dark conditions and, to a lesser degree, in constant conditions. The presence of a circadian melatonin rhythm in vitro in winter, although such a rhythm is absent in vivo in winter, suggests that pineal melatonin production is influenced by an extrapineal oscillator in the intact animal that may either positively or negatively modulate melatonin production in summer and winter, respectively.     

Effects of sex factors and hemispheric localization on the involvement of serotonin from the frontal cortex, striatum, and nucleus accumbens into the processing of novel and repeatedly presented information in rats

The effects of novel or relevant (a single exposure to experimental chamber) and irrelevant (20 exposures to experimental chamber) stimuli on the levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the frontal cortex, striatum, and nucleus accumbens in the left and right hemispheres were studied in male and female rats. It was found that 5-HT and 5-HIAA contents in the frontal cortex changed in response to neither relevant nor irrelevant stimuli. However, there were hemispheric difference in 5-HT and 5-HIAA in the frontal cortex of intact animals. The level of 5-HT in males and the level of 5-HIAA in females were higher in the left frontal cortex. In females, the level of 5-HIAA in the left striatum decreased in response to the novel stimulus. Sex differences in: a) 5-HT metabolism (increase in the level of 5-HIAA in males and increase in 5-HT in females) and b) lateralization (the striatal 5-HT metabolism in males changed bilaterally and only in the left hemisphere in females) were observed in reactions to irrelevant stimuli. Both in male and female rats, serotonin content in the nucleus accumbens changed only in response to the irrelevant stimuli. The 5-HT level increased in the left and right hemispheres independently of sex, but hemispheric difference was revealed only in females, in which the serotonin level was higher in the left nucleus accumbens. It is concluded that serotonergic neurotransmitter mechanisms are involved in hemispheric and sex differences in selective attention.