The Role of the Brain in the Regulation of Peripheral Organs-Noradrenaline Sources in Neonatal Rats: Noradrenaline Synthesis Enzyme Activity.

This work is aimed at studying the mechanisms of reciprocal humoral regulation of noradrenaline-producing organs in rats in the perinatal period of development. The activity of noradrenaline synthesis enzymes tyrosine hydroxylase and dopamine-beta-hydroxylase was measured in the brain and adrenal glands 48 and 72 h after the injection of immunotoxin (anti-dopamine-beta-hydroxylase-saporin) into the rat brain ventricles. It was shown that, 48 h after the immunotoxin injection into the brain, the activity of tyrosine hydroxylase in the brain decreased; however, 72 h after the injection it reached the control levels. This fact indicates that noradrenaline synthesis in the survived neurons increases. In the adrenal glands, 72 h after the immunotoxin injection into the brain, the activity of dopamine-beta-hydroxylase increased. This points to a compensatory increase in the rate of noradrenaline synthesis in the adrenal glands when the synthesis of noradrenaline in the brain is inhibited.

Characteristic of Dopamine-Producing System and Dopamine Receptors in the Suprachiasmatic Nucleus in Rats in Ontogenesis.

One of the features of the developing suprachiasmatic nucleus (SCN), the "biological clock" of the body, is the early expression of dopamine (DA) receptors in the absence of dopaminergic neurons as a source of DA. Only recently we showed that DA in SCN is synthesized together by nerve fibers containing only tyrosine hydroxylase (TH) and neurons containing only aromatic L-amino acid decarboxylase (AADC). This study was aimed to assess specific characteristics of the phenotype of TH-fibers in ontogenesis. For this purpose, PCR and immunohistochemical analysis of the expression of genes and proteins such as TH, AADC, vesicular monoamine transporter (VMAT), and receptors for DA (D1, D2) was performed. We have detected numerous TH-immunoreactive fibers in SCN of young and adult rats. VMAT was observed in some of them, which suggests vesicular storage of L-DOPA. Considering the key role of TH-fibers in cooperative synthesis of DA, we assumed the presence of their dopamine regulation. Using double immunolabeling, we showed that D1 and D2 are present in TH-fibers in adult rats, and only D1 in young rats. According to PCR, D1 and D2 are also expressed in neurons of SCN in adult rats and only D1 in young rats. Thus, it was shown for the first time that VMAT and D1 are coexpressed in TH-fibers synthesizing L-DOPA in SCN in young and adult rats, and also D2 receptors in adult rats, which suggests vesicular storage and dopamine regulation of L-DOPA secretion, respectively.

Estimation of Metabolism of Catecholamines in Peripheral Organs As an Indicator of Their Desympathization under the Influence of Neurotoxins.

Under conditions of the experimental model of Parkinson's disease at the preclinical (early) and clinical stage-injection of mice with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 5 and 10 weeks-the toxic effects of MPTP was evaluated (the content of catecholamines and their metabolites in the heart, which receives the most extensive sympathetic innervation, was determined). The obtained data indicated the beginning of desympathization of the heart at the preclinical stage of PD and its progression at the clinical stage of the disease.

Proteins of the Vesicular Cycle as a Marker of Neuroplasticity of Dopaminergic Neurons in the Substantia Nigra of the Brain.

Nigrostriatal dopaminergic neurons (DNs), involved in the regulation of motor function, are characterized by a high plasticity. Indeed, at the death of up to 50% of DNs in Parkinson's disease, the survived neurons provide normal regulation. This study was aimed to determine whether the vesicle cycle proteins, syntaxin Ia (Syn Ia), synaptotagmin I (Syt I), Rab5a, and complexins I and II (Cmpx I and II) are involved in the mechanisms of neuroplasticity in the substantia nigra, which mainly contains cell bodies and processes of the DNs. In the neurotoxic models of Parkinson's disease in mice, it was shown that, at the degeneration of up to 50% of DNs, the content of Syt I, Syn Ia, and Cmpх I and II, involved in vesicle exocytosis, does not change in the substantia nigra as a whole but is compensatorily increased in individual survived DNs. Thus, the data obtained in this study suggest that the impairment of motor behavior, which occurs at the death of half of the nigrostriatal DNs, is not caused by the impairment of the production of vesicle cycle proteins in the survived DNs.

The Role of Catecholamines in the Development of Pathological Retina Neovascularization in an Experimental Model of Retinopathy of Prematurity in Rats.

This work is dedicated to proving our hypothesis that catecholamines and their metabolites play a crucial role in the development of retinopathy of prematurity, which leads to progressive uncontrollable vascularization in the retina, leading to blindness. The study was performed in an animal model of retinopathy of prematurity, which was achieved by hyperoxygenation in rats on postnatal days 7, 14, 21, and 30. The content of catecholamines and their metabolites in the retina of rats was determined by high performance liquid chromatography with electrochemical detection. It was shown that, in the rats with retinopathy, the content of L-DOPA on days 21 and 30 was decreased as compared to the control, whereas the content of noradrenaline on day 14 life increased compared to the control. However, we did not observe changes in the content of dopamine in the experimental animals relative to the control in any period studied. Given the published data on the involvement of catecholamines in the regulation of vasculogenesis in the retina in normal state, our data on the changes in the catecholamine metabolism in the retina in the model of retinopathy of prematurity can be regarded as evidence of the important role of catecholamines in the pathogenesis of this severe disease.

The Role of the Brain in the Regulation of Peripheral Noradrenaline-producing Organs in Rats During Morphogenesis.

This work represents one part of our research project, in which we attempted to prove that a humoral regulation between noradrenaline-producing organs exist in the perinatal period. In this study, we used a rat model that allowed blocking the synthesis of noradrenalin in the brain and evaluated gene expression and protein levels of noradrenaline key synthesis enzymes such as tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) in peripheral noradrenaline-producing organs. As a result, we showed an increased gene expression of TH and DBH in adrenal glands. These data indicate that, if neonatal rat brain lacks the ability to produce noradrenaline, then the synthesis of noradrenaline in adrenal glands increased as a compensatory process, so that the concentration levels in blood are maintained at normal levels. This indicates that there is a humoral regulation between brain and adrenal glands, which is not fully understood yet.

Dopamine Synthesis as a Mechanism of Brain Plasticity in Nigrostriatal System Pathology.

Using an experimental Parkinson's disease model (symptoms develop in mice after the injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), we studied the characteristics of the synthesis of dopamine as a possible compensatory mechanism aimed at maintaining the dopamine level in the dopaminergic neurons that survived in this pathology. We found no correlation between the content and activity of tyrosine hydroxylase in the nigrostriatal system. The enzyme activity and the dopamine content showed unidirectional changes in the substantia nigra, but not in the striatum, which is apparently due to triggering other compensatory mechanisms.

General Sources of Dopamine As a Potential Morphogenic Factor in the Developing Striatum of Rats.

In the striatum of rats at different stages of development, we determined the content of tyrosine hydroxylase and aromatic L-amino acids decarboxylase using double immunohistochemical labeling and estimated the expression level of their transcripts by real-time PCR. We found that, in different periods of development, there are three sources of dopamine in the striatum of rats: bienzymatic nerve fibers (throughout ontogeny), bienzymatic neurons (appear on day 18 of embryonic development), and monoenzymatic neurons (in adult animals). Dopamine, which is synthesized in the striatal neurons in the prenatal period, may function as a morphogenetic factor.

[Endothelins and dopamine levels in tears for assessment of neurovascular disorders in glaucoma]. / Éndoteliny i dofamin v sleznoi zhidkosti v otsenke neirovaskuliarnykh narushenii pri glaukome.

PURPOSE: To estimate the possibility of detection of neurovascular ocular disorders in glaucoma by assessing the content of catecholamines and endothelins in lacrimal fluid. MATERIAL AND METHODS: The study included 47 patients with primary open-angle glaucoma (POAG). Tear eluate was analyzed by high performance liquid chromatography (HPLC) for catecholamines concentrations, and enzyme-linked immunoassay (ELISA) was used for evaluation of endothelins content. RESULTS: Endothelin-1 (ET-1) and big endothelin (bET) content in tears of patients with POAG was higher than in healthy controls. Concentration of dopamine (DA) in tears was lower and concentrations of L-dioxyphenylalanine and dihydroxyphenylacetic acid had a tendency for decrease. Noradrenaline content was equal in patients with POAG and controls. Adrenaline was not detected in any tear samples. CONCLUSION: Multidirectional changes of endothelins and DA levels in tears of patients with POAG was found. The increased concentration of ET-1 and its precursor bET promote vasoconstriction and decrease of aqueous humor outflow. The decrease of DA concentration is typical for neurodegenerative processes. Estimation of DA and endothelins concentrations in tears can enable early detection of neurovascular disorders in glaucoma patients and help evaluate their severity.

Cooperative Synthesis of Dopamine in Rat Mediobasal Hypothalamus as a Compensatory Mechanism in Hyperprolactinemia.

Dopamine (DA), synthesized in the mediobasal hypothalamus by dopaminergic neurons containing two enzymes of DA synthesis - tyrosine hydroxylase and decarboxylase of aromatic L-amino acids, or by monoenzymatic non-dopaminergic neurons containing one DA synthesis enzyme in cooperation, is known to have an inhibitory effect on prolactin secretion. Deterioration of this inhibitory control leads to an increase in prolactin concentration in the blood and to the development of hyperprolactinemia syndrome. In a rat model of hyperprolactinemia induced by administration of a neurotoxin causing degeneration of dopaminergic and noradrenergic neurons, the level of DA first decreases, leading to an increase in prolactin level (decompensation stage), while later both levels are restored to normal (compensation stage). However, the mechanism of such compensation is still not clear. The aim of the present study was to analyze whether the increase in cooperative synthesis of DA by monoenzymatic neurons during hyperprolactinemia is a manifestation of a compensatory mechanism representing a particular case of neuroplasticity. The level of cooperative synthesis in the hyperprolactinemia model and in the control was estimated as the level of synthesis of DA and L-dihydroxyphenylalanine (L-DOPA) - an intermediate product of DA synthesis, when L-DOPA transfer from neurons containing tyrosine hydroxylase into neurons containing aromatic L-amino acid decarboxylase is inhibited. The level of DA synthesis during the decompensation stage was not changed, while during the compensation stage it was lower than the control. Along with a reduction in DA level, during the compensation stage an increase in the extracellular L-DOPA level in the medium was detected. Thus, the compensation of DA deficiency after degeneration of dopaminergic neurons in the mediobasal hypothalamus is due to the increase in cooperative synthesis of DA by monoenzymatic neurons containing one of the complementary enzymes of the DA synthesis pathway.

Plasticity of Central and Peripheral Sources of Noradrenaline in Rats during Ontogenesis.

The morphogenesis of individual organs and the whole organism occurs under the control of intercellular chemical signals mainly during the perinatal period of ontogenesis in rodents. In this study, we tested our hypothesis that the biologically active concentration of noradrenaline (NA) in blood in perinatal ontogenesis of rats is maintained due to humoral interaction between its central and peripheral sources based on their plasticity. As one of the mechanisms of plasticity, we examined changes in the secretory activity (spontaneous and stimulated release of NA) of NA-producing organs under deficiency of its synthesis in the brain. The destruction of NA-ergic neurons was provoked by administration of a hybrid molecular complex - antibodies against dopamine-ß-hydroxylase associated with the cytotoxin saporin - into the lateral cerebral ventricles of neonatal rats. We found that 72 h after the inhibition of NA synthesis in the brain, its spontaneous release from hypothalamus increased, which was most likely due to a compensatory increase of NA secretion from surviving neurons and can be considered as one of the mechanisms of neuroplasticity aimed at the maintenance of its physiological concentration in peripheral blood. Noradrenaline secretion from peripheral sources (adrenal glands and the organ of Zuckerkandl) also showed a compensatory increase in this model. Thus, during the critical period of morphogenesis, the brain is integrated into the system of NA-producing organs and participates in their reciprocal humoral regulation as manifested in compensatory enhancement of NA secretion in each of the studied sources of NA under specific inhibition of NA production in the brain.

Gene expression and content of enzymes of noradrenaline synthesis in the rat organ of Zuckerkandl at the critical period of morphogenesis.

Gene expression and content of the key enzymes involved in the synthesis of noradrenaline-tyrosine hydroxylase and dopamine beta-hydroxylase-was evaluated in the organ of Zuckerkandl of rats in the critical period of morphogenesis. High levels of mRNA and protein of both enzymes in the perinatal period of development and their sharp decline on day 30 of postnatal development were detected. These data indicate that the synthesis of noradrenaline in the organ of Zuckerkandl is maximum during the critical period of morphogenesis and decreases during the involution of this paraganglion.

Molecular mechanisms of synthesis of noradrenaline as an inducer of development in the adrenal glands of rats in ontogenesis.

The level of gene expression and the protein content of tyrosine hydroxylase and dopamine ß-hydroxylase were determined. In the perinatal period of rats, when noradrenaline functions as a morphogenetic factor, the level of gene expression of these enzymes increased and the content of protein products of these genes was almost unchanged, indicating the difference in the regulatory mechanisms of their transcription and translation.

[Ratio of transcription factor PHF10 splice variants in lymphocytes as a molecular marker of Parkinson's disease].

Parkinson's disease (PD) is the second most common neurodegenerative disorder and causes degeneration of dopaminergic neurons in the nigrostriatal system of the brain. PHF10 is one of the most important regulatory subunits of the SWI/SNF chromatin-remodeling protein complex, which controls the gene function and chromatin state in neurons. Two alternative RHF10 isoforms, PHF10-P and PHF10-S, replace each other in the complex to change the target gene pattern. Expression of the PHF10-P and PHF10-S transcripts in the nigrostriatal system and their ratio in blood lymphocytes were found to change in a mouse model of early clinical stage of PD as compared with control mice. Changes in PHF10-S level were also observed in peripheral blood lymphocytes from patients with early clinical stage of PD. A ratio of the PHF10-P and PHF10-S transcripts in peripheral blood cells was assumed to provide a potential marker of early stage PD.

Gene expression of proteins of the vesicle cycle in dopaminergic neurons in modeling of Parkinson's disease.

It is assumed that one of the causes of the degeneration of dopaminergic neurons is the dysregulation of the vesicle cycle, which is ensured by a number of proteins including syntaxin I, synaptotagmin I, complexins I and II, and Rab5. It was shown that there is a compensatory increase in gene expression of proteins responsible for exocytosis at the preclinical stage of Parkinson's disease (PD) in the in substantia nigra (SN) in mice. Conversely, in the model of the clinical stage of PD, the decreases of gene expression of proteins responsible for exocytosis, endocytosis, and neuronal survival, which may be among the triggers of motor dysfunctions.

Gene expression of proteins of the vesicle cycle in the striatum and motor cortex under functional failure of nigrostriatal system.

Degeneration of dopaminergic neurons in the substantia nigra and the decrease in the dopamine level in the striatum lead to dysfunctions of motor behavior. This is accompanied by dysregulation of neuro-transmission in glutamatergic neurons of the motor cortex and GABA-ergic neurons of the striatum. It is shown that dysregulation of the gene expression of vesicle cycle proteins in neurons of the motor cortex occurs at an early (presymptomatic) stage of degeneration of the nigrostriatal system, and in more severe degeneration (symptomatic stage) the level of gene expression of vesicle cycle proteins in the striatum decreases.

Missing proof of cooperative synthesis of dopamine by non-dopaminergic neurons.

L-DOPA accumulation in the extracellular medium was detected when the transfer of L-DOPA from the neurons containing tyrosine hydroxylase to the neurons containing aromatic L-amino acid decarboxylase was blocked, under conditions of inhibition of the L-DOPA degradation enzyme. Thus, the missing proof confirming the existence of cooperative synthesis of dopamine by neurons non-dopaminergic was obtained.

Signal molecules during the organism development: Central and peripheral sources of noradrenaline in rat ontogenesis.

Using the method of high performance liquid chromatography with electrochemical detection, the age dynamics of the content of noradrenaline (NA) in the brain, adrenal gland, and the organ of Zuckerkandl in prenatal (18th and 21st days of embryogenesis) and early postnatal (3, 7, 15, and 30th days) periods of development was studied. The potential contribution of these organs to the formation of physiologically active concentration of noradrenalin in the blood was also assessed. The results suggest that, during the development of the organism, the activity of the sources of noradrenaline in the general circulation changes, which gives a reason to assume the existence of humoral interaction between NA-producing organs in the perinatal period of ontogenesis.

Secretory activity of the brain and peripheral organs: Spontaneous and stimulated release of noradrenaline in the ontogenesis of rats.

Spontaneous and K(+)-stimulated release of noradrenaline from the hypothalamus, adrenal gland, and organ of Zuckerkandl under their flowing incubation was investigated in the perinatal period of ontogenesis of rats. The results suggest that, during the investigated period of ontogenesis, adrenal glands are the main source of noradrenaline in the blood, whereas the contributions of the organ of Zuckerkandl and the brain are not as significant and change during this period.

Role of Adenohypophysotropic Neurohormones in Endocrine Paraadenohypophysial Regulation of Peripheral Target Organs in Rat Ontogeny.

We tested the hypothesis that brain-derived chemical stimuli contribute to direct endocrine regulation of peripheral organs during ontogeny before blood-brain barrier closure. Dopamine and gonadotropin-releasing hormone present in high concentration in peripheral blood only before blood-brain barrier closure were chosen as the chemical stimuli. It was shown than dopamine in concentrations equal to its level in the peripheral blood inhibits prolactin secretion in organotypic culture of the pituitary gland from newborn rats via specific receptors. Experiments on organotypic culture of neonatal rat testicles showed that gonadotropin-releasing hormone stimulates testosterone secretion via specific receptors. We proved that chemical stimuli entering common circulation from the brain before blood-brain barrier closure could exert direct endocrine effect on peripheral organs.