Determination of dopamine, noradrenaline, and adrenaline in Krebs-Henseleit solution by liquid chromatography coupled with tandem mass spectrometry and measurement of their basal release from Chelonoidis carbonaria aortae in vitro.

This study presented for the first time the development and validation of a sensitive method for quantification of dopamine, noradrenaline, and adrenaline in Krebs-Henseleit solution by LC-tandem mass spectrometry. Aliquots of 2.0 mL calibrators, quality controls, and samples of Krebs-Henseleit solution incubated with tortoise's aortic ring for 30 min were extracted by solid-phase extraction. Catecholamine separation was achieved on a 100 × 4.6 mm LiChrospher RP-8 column and the quantification was performed by a mass spectrometer equipped with an electrospray interface operating in positive ion mode. The run time was 4 min and the calibration curve was linear over the range of 0.1-20.0 ng/mL. The method was applied to the measurement of basal release of dopamine, noradrenaline, and adrenaline from the tortoise Chelonoidis carbonaria aortae in vitro. One aortic ring (30 mm) per tortoise (n = 5) was incubated for 30 min in a 5 mL organ bath filled with Krebs-Henseleit solution. The method demonstrated sensitivity, precision, and accuracy enough for its application in the measurement of basal release of these catecholamines from C. carbonaria aortic rings in vitro. The mean (standard deviation) concentrations of dopamine, noradrenaline, and adrenaline were 3.48 (2.55) ng/mL, 1.40 (0.57) ng/mL, and 1.87 (1.09) ng/mL, respectively.

Impact of the Chronic Omega-3 Fatty Acids Supplementation in Hemiparkinsonism Model Induced by 6-Hydroxydopamine in Rats.

Parkinson's disease (PD) is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra. The neuronal degeneration may result from the convergence of a number of different pathogenic factors, including apoptosis, excitotoxicity and oxidative stress. Many studies emphasize the importance of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) in vital processes such as maintenance of the properties of cell membranes and the participation in signal transduction and biodynamic activity of neuronal membranes. In this study, the protective effect of ω-3 PUFA administration on the 6-hydroxydopamine (6-OHDA) model of PD in rats was investigated. ω-3 PUFA (1.5 and 3.0 g/kg) was orally administered by gavage during 28 consecutive days to male Wistar rats. On the 4th day, hemiparkinsonism was induced through intrastriatal injection of 6-OHDA. On the 25th day, the animals were submitted to behavioural analysis. On the 28th day, after euthanasia, the brain areas were collected for neurochemical evaluation. ω-3 PUFAs (1.5 and 3.0 g/kg) restored monoamine and amino acid levels on the striatum from hemiparkinsonian rats, followed by reduction in the number of apomorphine-induced rotations and promotion of a partial locomotor recovery. In addition, ω-3 PUFAs (1.5 and 3.0 g/kg) decreased the lipid peroxidation levels and nitrite levels in the brain areas from hemiparkinsonian rats. Thus, this study suggests that supplementation with ω-3 PUFAs prevents behavioural and neurochemical disturbances induced by 6-OHDA, presenting a potential neuroprotective action.

Repeated methylphenidate administration during lactation reduces maternal behavior, induces maternal tolerance, and increases anxiety-like behavior in pups in adulthood.

Methylphenidate (MPD) is a dopamine uptake inhibitor and the most commonly prescribed drug for the treatment of attention-deficit/hyperactivity disorder in children. Several studies have shown that such stimulants as cocaine and amphetamine that are administered during gestation and lactation may disrupt maternal behavior. Also, MPD is used in lactation. Repeated MPD administration can induce either sensitization or tolerance. The aim of the present study was to investigate whether repeated MPD administration alters maternal behavior and promotes tolerance or sensitization in these females. The effects in adult offspring were also examined in models of anxiety. Methylphenidate (5mg/kg) was administered from lactation day 2 to 4, and maternal pup retrieval behavior was assessed. This treatment was continued until lactation day 7. At weaning, the dams received a challenge dose of MPD, and general activity was evaluated in the open field. Striatal monoamine and metabolite levels were also measured to determine whether this treatment promotes behavioral or biochemical plasticity. The long-term behavioral effects of MPD exposure were evaluated in pups in adulthood. The results showed an increase in the latency to retrieve the first, second, and third pups and a decrease in the number of dams that retrieved all pups. After a challenge dose of MPD, the dams exhibited a decrease in locomotion frequency, an increase in immobility duration in the open field, and a decrease in striatal serotonin levels. In pups, anxiety-like behavior increased in the light/dark box test. These results indicate that repeated MPD administration during early lactation impairs maternal behavior, likely by decreasing maternal motivation. Repeated MPD administration induced maternal tolerance at weaning after a challenge dose of MPD, suggesting the development of central nervous system plasticity. In pups, maternal exposure to MPD during early lactation induced long-term effects and increased anxiety-like behavior in adulthood.

Antidepressant-like behavioral, anatomical, and biochemical effects of petroleum ether extract from maca (Lepidium meyenii) in mice exposed to chronic unpredictable mild stress.

Maca has been consumed as a medical food in Peru for thousands of years, and exerts anxiolytic and antidepressant effects. Our present study aimed to evaluate the behavior and anatomical and biochemical effects of petroleum ether extract from maca (ME) in the chronic unpredictable mild stress (CUMS) model of depression in mice. Three different doses of maca extract (125, 250, and 500 mg/kg) were orally administrated in the six-week CUMS procedure. Fluoxetine (10 mg/kg) was used as a positive control drug. Maca extract (250 and 500 mg/kg) significantly decreased the duration of immobility time in the tail suspension test. After treatment with maca extract (250 and 500 mg/kg), the granule cell layer in the dentate gyrus appeared thicker. Maca extract (250 and 500 mg/kg) also induced a significant reduction in corticosterone levels in mouse serum. In mouse brain tissue, after six weeks of treatment, noradrenaline and dopamine levels were increased by maca extract, and the activity of reactive oxygen species was significantly inhibited. Serotonin levels were not significantly altered. These results demonstrated that maca extract (250 and 500 mg/kg) showed antidepressant-like effects and was related to the activation of both noradrenergic and dopaminergic systems, as well as attenuation of oxidative stress in mouse brain.

Effect of 2,4-dichlorophenoxyacetic acid on milk transfer to the litter and prolactin release in lactating rats.

The effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on brain monoamines and the serum level of hormones involved in milk synthesis and on the milk ejection reflex in rats were evaluated. Dams were treated with 2.5, 5, 15, 25, 50 or 70mg 2,4-D/kg bw according to two experimental designs: (a) through food from post partum day 1 (PPD 1) to PPD 16 and the respective control groups or (b) an unique i.p. injection on PPD 11. To measure milk ejection, the litter was separated from the mother at the 11th day of lactation during 8h, returned to their mothers and allowed to suckle for a period of 15min. The procedure was repeated on 3 consecutive days until the end of treatment. The change in litter weight during the suckling period was taken as a measure of the amount of milk ejected during this period. The dams' serum prolactin (PRL), oxytocin (OT) and growth hormone levels were determined by radioimmunoassay. Both treatment regimens produced a dose-dependent decrease in the amount of milk ejected and circulating PRL and OT secreted in response to the suckling stimulus. Administration of OT before returning the pups restored the milk ejection, indicating no impairment in the capacity of the mammary gland to produce and secrete milk. In addition, dopamine levels were increased by the 2,4-D treatments in arcuate nucleus (ArN) and anterior lobe of pituitary gland (AL), while serotonin level was drastically decreased in ArN. 2,4-D treatment increased both calcium-dependent and calcium-independent nitric oxide synthase (NOS) activities in ArN. These results suggest that 2,4-D inhibits the suckling-induced hormone release, milk transfer to the litter at the central level, through a stimulation of hypothalamic NOS and dopamine and by an inhibition of hypothalamic serotonin transmission.

Effects of maternal exposure to an aromatase inhibitor on sexual behaviour and neurochemical and endocrine aspects of adult male rat.

The present study examined the effects of letrozole exposure during brain sexual differentiation on endocrine, behavioural and neurochemical parameters in male rat descendants. Pregnant female rats received 1 mg kg(-1) day(-1) letrozole or vehicle by oral gavage on gestational Days 21 and 22. Exposure to letrozole reduced anogenital distance in males on postnatal Day (PND) 22. At adulthood (PND 75), plasma testosterone levels and hypothalamic dopaminergic activity were increased, but sexual competence was impaired, because fewer successful sexual behaviours (mount, intromission and principally ejaculation) were observed. The impairment of reproductive function by prenatal exposure to an aromatase inhibitor reinforces the importance of adequate oestrogenic activity during perinatal sexual differentiation for complete masculinisation of the hypothalamus.

Rats exposed to Solanum lycocarpum fruit in utero and during lactation: neurochemical, behavioral and histopathological effects.

Solanum lycocarpum St. Hil (Solanaceae) is a native shrub very common in the Brazilian savannah. This plant contains steroidal glycoalkaloids that can be transformed into an intermediate for steroidal drug production. In this way, it is very possible that these glycoalkaloids and its aglycone, once in the body by ingestion of S. lycocarpum fruits, may act by disrupting the endocrine system. Because its fruits may be consumed by pregnant animals in the fields, the present study determined the possible toxic effects of exposure to S. lycocarpum fruit (10% added in the diet) from gestation day (GD) 6 to postnatal day (PND) 07 in rat dams. The unripe fruits contained 0.6% of solamargine and 0.9% of solasonine. S. lycocarpum, 10% in the diet, during gestation and the beginning of lactation reduced intrauterine growth. In addition, 20% of the treated dams showed some dead pups at birth. Reduced body weight was observed from birth through adulthood in male and female offspring exposed to 10% S. lycocarpum unripe fruits. During adulthood, female offspring showed impaired sexual behavior and male offspring showed prominent degeneration of testis germinative cells, characterized by a reduced number of germ cells and vacuolation. Also, the exposed offspring showed reduced hypothalamic norepinephrine (NOR), vanillylmandelic acid (VMA), 3-methoxy-4-hydrophenylglycol (MHPG) and homovanillic acid (HVA) levels, and reduced striatum NOR, HVA, VMA, MHPG, dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5-HIAA) levels. These results suggest that the fruit may act as an estrogen, with a long-term effect, impairing the receptive lordosis behavior of female offspring and promoting testis abnormalities in male offspring at adulthood. Finally, it appears to disrupt brain organization since important central monoamine level alterations were also observed.

Amperometric biosensor based on horseradish peroxidase for biogenic amine determinations in biological samples.

An amperometric biosensor for total biogenic amine determinations, using a carbon paste electrode modified with horseradish peroxidase (HRP) enzyme is described. The HRP immobilization on graphite was made using bovine serum albumin, carbodiimide and glutaraldehyde. The biosensor response was optimized using serotonin and it presented the best performance in 0.1 mol l(-1) phosphate buffer (pH=7.0) containing 10 micromol l(-1) of hydrogen peroxide. Under optimized operational conditions at -50 mV versus SCE, a linear response range from 40 to 470 ng ml(-1) was obtained. The detection limit was 17 ng ml(-1) and the response time was 0.5s. The proposed sensor presented a stable response during 4h under continuous monitoring. The difference of the response between six sensor preparations was <2%. The sensor was applied in the determination of total biogenic amines (neurotransmitters) in rat blood samples with success, obtaining a recovery average of 102%.

Effects of Ipomoea carnea aqueous fraction intake by dams during pregnancy on the physical and neurobehavioral development of rat offspring.

The effects of daily prenatal exposure to 0.0, 0.7, 3.0 and 15.0 mg/kg of the aqueous extract (AQE) of Ipomoea carnea dried leaves on gestational days 5-21 were studied in rat pups and adult offspring. The physical and reflex developmental parameters, open-field, plus-maze, social interaction, forced swimming, catalepsy and stereotyped behaviors, as well as striatal, cortical and hypothalamic monoamine levels (at 140 days of age) were measured. Maternal and offspring body weights were unaffected by exposure to the different doses of the AQE. High postnatal mortality, smaller size at Day 1 of life, reversible hyperflexion of the carpal joints and delay in the opening of both ears and in negative geotaxis were observed in the offspring exposed to the higher dose of AQE. At 60 and 90 days of age, open-field locomotion frequency was quite different between 0.0 and animals treated with 0.7 and 3.0 mg/kg AQE. No changes were observed in the plus-maze, social interaction, forced swimming, catalepsy, stereotyped behavior and central nervous system monoamines concentrations. Dams treated with the higher AQE dose showed severe cytoplasmic vacuolation in liver, kidney, pancreas and thyroid tissues, in contrast to the mild vacuolation observed in the other experimental groups. No alterations were observed in the histopathological study of the offspring of all experimental groups at 140 days of age. During adulthood, behavior was not modified in offspring exposed to the higher dose of AQE as well as no changes occurred in central nervous system neurotransmitters. The present data show that the offspring development alterations were not severe enough to produce behavioral and central monoamine level changes.

Effects of taurine deficiency and chronic methanol administration on rat retina, optic nerve and brain amino acids and monoamines.

A chronic methanol (MeOH) intoxication scheme (2 g/kg/day ip for 2 weeks) was carried out in Sprague-Dawley rats, previously depleted of folates with methotrexate (MTX). beta-Alanine (beta-Ala), 5%, was also administered to some animals in the drinking water. Amino acids were determined in plasma, retina, optic nerve, hippocampus and posterior cortex by HPLC with fluorescence detection and monoamines in retina, hippocampus and posterior cortex by electrochemical detection. Beta-Ala administration reduced taurine (Tau) levels in plasma, hippocampus and posterior cortex, but not in retina and optic nerve. Aspartate (Asp) concentration in the optic nerve was increased in MTX-MeOH treated animals, and the administration of beta-Ala did not modify this elevation. The association of beta-Ala with MTX-MeOH produced an increase of threonine, and a decrease of 5-hydroxytryptamine (5-HT) in the retina without modifying 5-hydroxyindoleacetic acid, whereas in the hippocampus an elevation of asparagine was observed. We conclude that, in the retina, beta-Ala in combination with MTX-MeOH increased serotonin and decreased dopamine (DA) turnover rate, and resulted in changes in the amino acid balance, that could affect glycinergic activity. On the other hand, in the hippocampus, Asp metabolism could be affected by Tau depletion with beta-Ala.

Effect of single and repeated methamphetamine treatment on neurotransmitter release in substantia nigra and neostriatum of the rat.

The main purpose of this study was to characterize the initial neurotransmission cascade elicited by methamphetamine, analysing simultaneously with in vivo microdialysis monoamine, amino acid and neuropeptide release in substantia nigra and neostriatum of the rat. The main effect of a single systemic dose of methamphetamine (15 mg/kg, subcutaneously) was an increase in dopamine levels, both in substantia nigra ( approximately 10-fold) and neostriatum ( approximately 40-fold), accompanied by a significant, but lesser, increase in dynorphin B ( approximately two-fold, in both regions), and a decrease in monoamine metabolites. A similar effect was also observed after local administration of methamphetamine (100 microm) via the microdialysis probes, but restricted to the treated region. In other experiments, rats were repeatedly treated with methamphetamine or saline, with the last dose administered 12 h before microdialysis. Dopamine K+-stimulated release was decreased following repeated methamphetamine administration compared with that following saline, both in the substantia nigra (by approximately 65%) and neostriatum (by approximately 20%). In contrast, the effect of K+-depolarization on glutamate, aspartate and GABA levels was increased following repeated administration of methamphetamine. In conclusion, apart from an impairment of monoamine neurotransmission, repeated methamphetamine produces changes in amino acid homeostasis, probably leading to NMDA-receptor overstimulation.

Influence of different antidepressant drugs on the effect of chronic variable stress on restraint-induced dopamine release in frontal cortex.

The aim of this study was to evaluate the influence of an early chronic variable stress procedure (CVS) associated or not with repeated administration of various antidepressants on cortical restraint-induced dopamine (DA) release in vivo. Animals were subjected to the CVS schedule and one day after submitted to persistent administration with vehicle, desipramine (DMI, 10 mg/kg, i.p.), fluoxetine (FLU, 10 mg/kg, i.p.) or phenelzine (PHE; 10 mg/kg, i.p.) and later on exposed to a 60-min restraint period. In addition, we also explored the effect of acute administration of these antidepressants on cortical DA overflow in response to restraint in CVS treated rats. A higher increase in cortical DA release in response to restraint was observed in CVS animals as compared with those without previous CVS. Persistent, but not acute, administration with DMI, FLU and PHE blocked the sensitized output induced by restraint following CVS exposure.