This thesis deals with the development and optimization of a method for the determination of tryptophan and its metabolites - serotonin and melatonin - in plant material, in grapevine, during one analysis. It uses a high-pressure liquid chromatography. The theoretical part is about tryptophan, its metabolism and basic properties of its metabolites - serotonin and melatonin. Their occurrence in wine is also discussed. Analytical techniques by which these analytes can be determined are also provided. Then information about modern stationary phases, that are suitable for this species, is included. The experimental part consists of optimization of the method, measurement of calibration dependences and measurement of real samples. It is measured by the method of reverse phase chromatography. As first stationary phase it is used a C18 column with core-shell packing, second is a BEH Phenyl column. The mixture of 10 mM acetate buffer (pH = 4.5) and methanol is used as the mobile phase. For detection UV at wavelength 254 nm is used, then for greater sensitivity mass detectionis is used. The basic conditions for the experiment have been set. At the beginning of the analysis, the mobile phase contains 95 % (v/v) buffer and 5 % (v/v) methanol. Then the methanol content is linearly increased to 80 % (v/v) from...

The naturally occuring ergot alkaloids of the fungus, Claviceps purpurea, and their many derivatives have been of neuropharmacological interest for many years because of their ability to affect peripheral and central adrenergic and serotonergic systems. More recently, selected compounds such as lergotile (2-chloro-6-methyl ergoline-8-beta acetonitrile) and and bromocriptine (2-bromo-alpha-ergocryptine), have been given additional attention due to their possible therapeutic potential in the treatment of parkinson’s disease, acromegaly and other disorders. There have been considerable data published attempting to establish the mechanism(s) whereby the ergot compounds exert their effects. A large portion of these experiments involves the interaction of ergot compounds with dopaminergic systems. This is a logical course of study, since many of the actions of the ergot compounds mimic the actions of compounds known to affect dopaminergic neurons, e.g. antagonists such as the phenothiazines and butyrophenones and agonists such as levodopa and apomorphine. In the last decade, much attention also has been focused on the role of serotonin (5-hydroxytryptamine) in the mediation of dopaminergic systems. There have been many conflicting reports published as to the role of serotonin but it is still uncertain whether or not serotonin does indeed play a role. The present study investigates two dopaminergic effects of the standard dopamine agonist apomorphine and the ergoline lergotrile and the similarities or differences that exist when serotonergic function is altered.

The ontogenic destruction of dopamine (DA) neurons in rat brain is associated with supersensitization of DA D1 receptors. This effect is attenuated when rats are cotreated in ontogeny with the serotonin (5-HT) neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT). In an attempt to determine whether 5-HT fibers might have a similar modulatory role on the sensitivity of the DA D2 receptor complex, we pretreated rats with desipramine HCl (20 mg/kg IP, base), 1 h before the DA neurotoxin, 6-hydroxydopamine (6-OHDA; 134 μg ICV, base) and/or 5,7-DHT (75 μg ICV) and/or vehicle. At about 3 months after birth dose-effect curves for quinpirole-induced oral activity were constructed for each group of rats. We found that quinpirole, an agonist for the DA D2 receptor complex, produced a dose-related increase in oral activity in all groups of rats. After a 200 μg/kg dose of quinpirole HCl, however, neonatal 5,7-DHT-lesioned rats had a peak oral response of 54.4 ± 5.1 (mean and SEM) vs. 22.6 ± 4.8 for control rats (p < 0.01). In neonatal 6-OHDA-lesioned rats this dose of quinpirole increased oral activity to 36.8 ± 5.8 oral movements (p < 0.05 vs. control). In rats lesioned with both 5,7-DHT and 6-OHDA, the oral response was not different from control. The enhanced oral response to quinpirole in 5,7-DHT-lesioned rats was attenuated by spiperone, an antagonist for the DA D2 receptor complex. These findings are believed to be the first to demonstrate that receptors of the DA D2 complex become sensitized after ontogenic injury to 5-HT fibers. This effect is opposite to the attenuated sensitivity of DA D1 receptors in rats with a similar 5-HT lesion.

Dopamine (DA) neurons are implicated in the hyperlocomotion of neonatal 6-hydroxydopamine (6-OHDA)-lesioned rats, an animal model of attention deficit hyperactivity disorder (ADHD). Because serotonin (5-HT) neurons mediate some DA agonist effects, we investigated the possible role of 5-HT neurons on locomotor activity. Rats were treated at 3 days after birth with vehicle or 6-OHDA (134 μg ICV; desipramine pretreatment, 20 mg/kg IP, 1 h), and at 10 weeks with vehicle or 5,7-dihydroxytryptamine (5,7-DHT; 75 μg ICV; pretreatment with desipramine and pargyline, 75 mg/kg IP, 30 min), to destroy DA and/or 5-HT fibers. Intense spontaneous hyperlocomotor activity was produced in rats lesioned with both 6-OHDA and 5,7-DHT. Locomotor time in this group was 550 ± 17 s in a 600 s session, vs. 127 ± 13 s in the 6-OHDA group and <75 s in 5,7-DHT and intact control groups (p < 0.001). Oral activity dose-effect curves established that 5,7-DHT attenuated DA D1 receptor supersensitivity and further sensitized 5-HT2c receptors. Acute treatment with dextroamphetamine (0.25 mg/kg SC) reduced locomotor time in 6-OHDA+5,7-DHT-lesioned rats to 76 ± 37 s (p < 0.001). Striatal DA was reduced by 99% and 5-HT was reduced by 30% (vs. 6-OHDA group). Because combined 6-OHDA (to neonates) and 5,7-DHT (to adults) lesions produce intense hyperlocomotion that is attenuated by amphetamine, we propose this as a new animal model of ADHD. The findings suggest that hyperactivity in ADHD may be due to injury or impairment of both DA and 5-HT neurons.

The classic rodent model of Parkinson’s disease (PD) is produced by unilateral lesioning of pars compacta substantia nigra (SNpc) in adult rats, producing unilateral motor deficits which can be assessed by dopamine (DA) D2 receptor (D2-R) agonist induction of measurable unilateral rotations. Bilateral SNpc lesions in adult rats produce life-threatening aphagia, adipsia, and severe motor disability resembling paralysis-a PD model that is so compromised that it is seldom used. Described in this paper is a PD rodent model in which there is bilateral 99% loss of striatal dopaminergic innervation, produced by bilateral intracerebroventricular or intracisternal 6-hydroxydopamine (6-OHDA) administration to perinatal rats. This procedure produces no lethality and does not shorten the life span, while rat pups continue to suckle through the pre-weaning period; and eat without impairment post-weaning. There is no obvious motor deficit during or after weaning, except with special testing, so that parkinsonian rats are indistin-guishable from control and thus allow for behavioral assessments to be conducted in a blinded manner. L-DOPA (L-3,4-dihydroxyphenylalanine) treatment increases DA content in striatal tissue, also evokes a rise in extraneuronal (i.e.,in vivo microdialysate) DA, and is able to evoke dyskinesias. D2-R agonists produce effects similar to those of L-DOPA. In addition, effects of both D1-and D2-R agonist effects on overt or latent receptor supersensitization are amenable to study. Elevated basal levels of reactive oxygen species (ROS), namely hydroxyl radical, occurring in dopaminergic denervated striatum are suppressed by L-DOPA treatment. Striatal serotoninergic hyperinnervation ensuing after perinatal dopaminergic denervation does not appear to interfere with assessments of the dopaminergic system by L-DOPA or D1-or D2-R agonist challenge. Partial lesioning of serotonin fibers with a selective neurotoxin either at birth or in adulthood is able to eliminate sero-toninergic hyperinnervation and restore the normal level of serotoninergic innervation. Of all the animal models of PD, that produced by perinatal 6-OHDA lesioning provides the most pronounced destruction of nigrostriatal neurons, thus representing a model of severe PD, as the neurochemical outcome resembles the status of severe PD in humans but without obvious motor deficits.

Chronic stress and dysfunction of the serotonergic system in the brain have been considered two of the major risks for development of depression. In this study, adult Fischer 344 rats were subjected to a regimen of chronic social defeat (CSD). To mimic stressful conditions, some rats were not exposed to CSD, but instead treated with corticosterone (CORT) in oral solution while maintained in their home cage. Protein levels of the serotonin transporter (SERT) in the dorsal raphe nucleus (DRN), hippocampus, frontal cortex, and amygdala were examined by Western blotting or immunofluorescence staining. The results showed that CSD up-regulated SERT protein levels in the DRN, hippocampus, frontal cortex, and amygdala regions. This up-regulation was abolished or prevented by adrenalectomy, or treatment with antagonists of corticosteroid receptors mifepristone and spironolactone, alone or in combination. Similarly, up-regulated SERT protein levels in these brain regions were also observed in rats treated with oral CORT ingestion, which was analogously prevented by treatment with mifepristone and spironolactone. Furthermore, both CSD- and CORT-induced up-regulation of SERT protein levels in the DRN and three brain regions were attenuated by simultaneous treatment with fluoxetine, an antidepressant that specifically inhibits serotonin reuptake. The results indicate that up-regulation in SERT protein levels in the DRN and forebrain limbic structures caused by CSD regimen was mainly motivated by CORT through corticosteroid receptors. The present findings demonstrate that chronic stress is closely correlated with the serotonergic system by acting on the regulation of the SERT expression in the DRN and its projection regions, which may contribute to the development of depression. Chronic stress and dysfunction of the serotonergic system are etiologically related to depression. In an attempt to explore their interaction, we found that chronic social defeat upregulated expression of serotonin transporter in the DRN and the projection regions, which may induce an alteration of serotonin transformation in the brain. This interaction may account for the development of this disease.

In this study, p-chlorophenylalanine (pCPA), an inhibitor of tryptophan hydroxylase (the rate limiting enzyme of serotonin synthesis), was used to reduce serotonin (5HT) levels during early development in zebrafish embryos. One day old dechorionated embryos were treated with 25μM pCPA for 24h and subsequently rescued. Immunohistological studies using a 5HT antibody confirmed that 5HT neurons in the brain and spinal cord were depleted of transmitter by 2days post fertilization (dpf). Twenty four hours after pCPA exposure embryos were unable to burst swim and were nearly paralyzed. Movement began to improve at 4dpf, and by 7dpf, larvae exhibited swimming activity. Rescued larvae continued to grow in rostrocaudal length over 5days post-rescue, but their length was always 16-21% below controls. Surprisingly, both groups displayed the same number of myotomes. To examine whether hypertonicity of myotomes in treated embryos played a role in their shorter rostrocaudal lengths, 1dpf embryos were exposed to a combination of 25μM pCPA and 0.6mM of the sodium channel blocker ethyl 3-aminobenzoate methanesulfonate (MS-222). After a 24hour exposure, the embryos exhibited the same rostrocaudal length as control embryos suggesting that myotome hypertonicity plays a major role in the decreased axial length of the treated larvae. In addition, pCPA treated 2dpf embryos exhibited abnormal notochordal morphology that persisted throughout recovery. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to determine the relative levels of the serotonin 1A receptor (5HT 1A) transcript and the serotonin transporter (SERT) transcript in the brain and spinal cord of control and treated embryos. Transcripts were present in both brain and spinal cord as early as 1dpf and reached maximal concentrations by 3dpf. Embryos treated with pCPA demonstrated a decrease in the concentration of 5HT 1A transcript in both brain and spinal cord. While SERT transcript levels remained unaffected in brain, they were decreased in spinal cord. Five days subsequent to pCPA rescue, 5HT 1A transcript concentrations remained decreased in brain while SERT transcript levels were elevated in both regions. These findings suggest that reduction of 5HT during early zebrafish development may have an adverse effect on body length, notochordal morphology, locomotor behavior, and serotonin message-related expression.

For more than 50 years, heavy metal exposure during pre- or post-natal ontogeny has been known to produce long-lived hyperactivity in rodents. Global brain injury produced by neonatal hypoxia also produced hyperactivity, as did (mainly) hippocampal injury produced by ontogenetic exposure to X-rays, and (mainly) cerebellar injury produced by the ontogenetic treatments with the antimitotic agent methylazoxymethanol or with polychlorinated biphenyls (PCBs). More recently, ontogenetic exposure to nicotine has been implicated in childhood hyperactivity. Because attention deficits most often accompany the hyperactivity, all of the above treatments have been used as models of attention deficit hyperactivity disorder (ADHD). However, the causation of childhood hyperactivity remains unknown. Neonatal 6-OHDA-induced dopaminergic denervation of rodent forebrain also produces hyperactivity - and this model, or variations of it, remain the most widely-used animal model of ADHD. In all models, amphetamine (AMPH) and methylphenidate (MPH), standard treatments of childhood ADHD, typically attenuate the hyperactivity and/or attention deficit. On the basis of genetic models and the noted animal models, monoaminergic phenotypes appear to most-closely attend the behavioral dysfunctions, notably dopaminergic, noradrenergic and serotoninergic systems in forebrain (basal ganglia, nucleus accumbens, prefrontal cortex). This paper describes the various pharmacological models of ADHD and attempts to ascribe a neuronal phenotype with specific brain regions that may be associated with ADHD.

Substance abuse is a frequent comorbid condition with other psychiatric disorders including schizophrenia and depression. These disorders may share a common substrate at the neurotransmitter or neurocircuit level. One candidate is hypofunction of the glutamate system. Several lines of evidence suggest that N-methyl-D-aspartate (NMDA) receptors may hypofunction in schizophrenia. Thus, NMDA receptor antagonists are schizophrenogenic; postmortem and imaging results point to reduced NMDA receptor function in schizophrenic brains; a number of genes that have been linked to schizophrenia code for proteins that influence NMDA function; and there is preliminary evidence that pro-NMDA drugs may be therapeutic in the treatment of schizophrenia. One of the most effective therapeutics for the treatment of substance abuse in schizophrenic people is clozapine, and clozapine may act at the glycine modulatory site to enhance NMDA receptor function. This preliminary line of evidence may link schizophrenia and drug abuse to a common neurochemical base, subnormal NMDA receptor function. People with schizophrenia and drug abusers similarly show deficits in tasks known to be sensitive to ventromedial prefrontal cortical damage, and both groups show decreased activation in the ventral striatum during reward anticipation in functional magnetic resonance imaging studies. These observations implicate common prefrontal cortical-striatal circuits and their modulation by hippocampal projections in schizophrenia and substance abuse. Withdrawal from substance abuse and depression both have been linked to changes in the function of several neurotransmitters including serotonin, dopamine and glutamate. These findings suggest possible common substrates and novel therapeutic approaches. Further studies are needed to fully characterize the neurocircuits and transmitters involved in various psychiatric disorders and their possible common elements in comorbid drug abuse.

This study examines the effects of the selective serotonin reuptake inhibitor (SSRI), fluoxetine (PROZAC), on the ontogeny of spontaneous swimming activity (SSA) in developing zebrafish. The development of zebrafish motor behavior consists of four sequential locomotor patterns that develop over 1-5 days post fertilization (dpf), with the final pattern, SSA, established at 4-5 dpf. In stage specific experiments, larvae were exposed to 4.6 μM fluoxetine for 24 h periods beginning at 24 h post fertilization (hpf) and extending through 5 dpf. From 1-3 dpf, there was no effect on SSA or earlier stages of motor development, i.e., spontaneous coiling, evoked coiling and burst swimming. Fluoxetine exposure at 3 dpf for 24 h resulted in a transient decrease in SSA through 7 dpf with a complete recovery by 8 dpf. Larvae exposed to 4.6 μM fluoxetine for 24 h on 4 or 5 dpf showed a significant decrease in SSA by day 6 with no recovery through 14 dpf. Although SSA was significantly affected 24 h after fluoxetine exposure, there was little or no effect on pectoral fin movement. These results demonstrate both a stage specific and a long term effect of 4.6 μM fluoxetine exposure in 4 and 5 dpf larvae. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to determine the relative levels of a serotonin transporter protein (SERT) transcript and the serotonin 1A (5-HT1A) receptor transcript in developing embryos/larvae over 1-6 dpf. Both transcripts were present at 24 hpf with the relative concentration of SERT transcript showing no change over the developmental time range. The relative concentration of the 5-HT1A receptor transcript, however, showed a two-tiered pattern of concentration. RT-PCR was also used to detect potential changes in the SERT and 5-HT1A receptor transcripts in 6 dpf larvae after a 24 h exposure to 4.6 μM fluoxetine on 5 dpf. Three separate regions of the CNS were individually analyzed, two defined brain regions and spinal cord. The two brain regions showed no effect on transcript levels subsequent to fluoxetine exposure, however, the spinal cord showed a significant decrease in both transcripts. These results suggest a correlation between decreased concentration of SERT and 5-HT1A receptor transcripts in spinal cord and decreased SSA subsequent to fluoxetine exposure.