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.

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.

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.

The Effect of Dopamine Secreted by the Brain into the Systemic Circulation on Prolactin Synthesis by the Pituitary gland in Ontogenesis.

This research was aimed at studying the brain's endocrine function in ontogenesis. It has been previously shown in our laboratory that the brain serves as the source of dopamine in the systemic circulation of rats prior to the formation of the blood-brain barrier. This paper provides direct evidence that dopamine secreted by the brain directly into the systemic circulation in this period of ontogenesis has an inhibitory effect on prolactin secretion by pituitary cells. These results provide the basis for a fundamentally new understanding of the brain's role in the neuroendocrine regulation of the development and function of peripheral target organs and, particularly in this study, the pituitary gland.

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.

[Reciprocal Humoral Regulation of Endocrine Noradrenaline Sources in Perinatal Development of Rats].

The goal of the present study was to verify our hypothesis of humoral interaction between the norepinephrine secreting organs in the perinatal period of ontogenesis that is aimed at the sustaining of physiologically active concentration of norepinephrine in blood. The objects of the study were the transitory organs, such as brain, organ of Zuckerkandl, and adrenals, the permanent endocrine organ of rats that releases norepinephrine into the bloodstream. To reach this goal, we assessed the adrenal secretory activity (norepinephrine level) and activity of the Zuckerkandl's organ under the conditions of destructed noradrenergic neurons of brain caused by (1) their selective death induced by introduction of a hybrid molecular complex, which consisted of antibodies against dopamine-ß-hydroxylase (DBH) conjugated with saporin cytotoxin (anti-DBH-saporin) into the lateral brain ventricles of neonatal rats; and (2) microsurgical in utero destruction of embryo's brain (in utero encephalectomy). It was observed that 72 h after either pharmacological or microsurgical norepinephrine synthesis deprivation in the newborn rat's brain, the level of norepinephrine was increased in adrenals and, conversely, decreased in the Zuckerkandl's organ. Therefore, the experiments with models of chronical inhibition of norepinephrine synthesis in prenatal and early postnatal rat's brain revealed changes in the secretory activity of peripheral norepinephrine sources. This, apparently, favors the sustaining of physiologically active norepinephrine level in the bloodstream.

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.

Neurons expressing individual enzymes of dopamine synthesis in the mediobasal hypothalamus of adult rats: functional significance and topographic interrelations.

Besides dopaminergic (DA-ergic) neurons having all enzymes of DA synthesis, tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC), "monoenzymatic" neurons expressing only one of them were found in the brain, mostly in the mediobasal hypothalamus (MBH). The aim of this study was to test our hypothesis that DA is synthesized by monoenzymatic neurons, i.e. l-3,4-dihydroxyphenylalanine (l-DOPA), which produced in the monoenzymatic TH neurons is transported in the monoenzymatic AADC neurons for DA synthesis. Incubation of MBH in Krebs-Ringer solution with l-leucine, a competitive inhibitor of l-DOPA uptake, was used to prevent a hypothetical l-DOPA capture into AADC-containing neurons. Incubation of the substantia nigra containing DA-ergic neurons under the same conditions served as the control. According to our data, the l-leucine administration provoked a decrease of DA concentration in MBH and in the incubation medium but not in the substantia nigra and respective incubation medium, showing a decrease of cooperative synthesis of DA in MBH. This conclusion was supported by an observation of higher concentration of l-DOPA in the incubation medium under perfusion of MBH with Krebs-Ringer solution containing tolcapone, an inhibitor of catechol-O-methyltransferase, and l-leucine than under perfusion with the same solution, but without l-leucine. Functional interaction between monoenzymatic TH and AADC neurons was indirectly confirmed by finding in electron microscopy their close relations in MBH. Besides monoenzymatic AADC neurons, any AADC-possessing neurons, catecholaminergic and serotoninergic, apparently, could participate in DA synthesis together with monoenzymatic TH neurons. This idea was confirmed by the observation of close topographic relations between monoenzymatic TH neurons and those containing both enzymes, i.e. DA-ergic, noradrenergic or adrenergic. Thus, monoenzymatic neurons possessing TH or AADC and being in close topographic relations can synthesize DA in cooperation.

[Development of serotonergic neurons of dorsal raphe nuclei in mice with knockout of monoamine oxidase A and 5-HT1A and 5-HT1B autoreceptor].

The morphological changes in the development of serotonergic neurons of the dorsal raphe nuclei in the medulla oblongata was studied by immunocytochemistry in mice with knockout of 1A and 1B serotonin autoreceptors as well as monoamine oxidase A. Serotonin autoreceptors regulate electric activity of serotonergic neurons as well as the synthesis and release of the neurotransmitter, while monoamine oxidase A catalyzes its degradation. These genetic modifications proved to have no effect on the number of serotonergic neurons in the medulla oblongata but induced morphofunctional changes. Decreased cell size and increased intracellular serotonin level were observed in the case of monoamine oxidase A deficiency, while excessive cell size and decreased intracellular serotonin level were observed in the case of autoreceptor deficiency. The data obtained confirm the hypothesis of autoregulation of serotonergic neurons in development.

[Development of central and peripheral serotonin-producing systems in rats in ontogenesis].

The work deals with study of development of central and peripheral serotonin-producing systems in rat ontogenesis before and after formation of the blood-brain barrier. By the method of highly efficient liquid chromatography it has been shown that the serotonin level in peripheral blood before formation of the blood-brain barrier (in fetuses and neonatal rats) is sufficiently high for realization of physiological effect on target cells and organs. At the period of formation of the blood-brain barrier the serotonin level in brain sharply rises, whereas the serotonin concentration and amount in plasma and duodenum increase insignificantly. Completion of formation of the blood-brain barrier is accompanied by a significant increase of the serotonin content in duodenum, probably for maintenance of the high serotonin level in blood. To evaluate secretory activity, the mean rate of daily increment of the serotonin in the studied tissues was calculated. In brain, this parameter was maximal at the period of formation of the blood-brain barrier and then sharply fell, whereas in duodenum it rose markedly after completion of the barrier formation. In plasma this parameter decreased statistically significantly at the period of formation of the blood-brain barrier - from the 4th to the 16th postnatal days. This allows thinking that brain before formation of the blood-brain barrier is a most important source of serotonin in peripheral blood.

[Brain is a source of blood serotonin in rats during perinatal development].

The aim of this study was to test our hypothesis that the brain functions as an endocrine organ before the blood-brain barrier is formed. A model of drug-inhibited serotonin synthesis in the brain using a single stereotactic administration of p-chlorophenylalanine, an inhibitor of serotonin synthesis, was developed. The inhibitor dose inducing the maximum effect in the brain and no effect on serotonin synthesis in the periphery was experimentally selected. The concentration of serotonin and its metabolites (5-hydroxytryptophan and 5-hydroxy-indoleacetic acid) was studied by high performance liquid chromatography in the brain, duodenum, and blood (separately in plasma and platelets). The optimal p-chlorophenylalanine dose (200 mg/kg) was shown to induce a sharp decrease in the brain level of serotonin (70%), a moderate decrease in plasma (16%) and platelets (26%), and an insignificant decrease in the duodenum (12%). At the same time, this dose did not decrease the 5-hydroxytryptophan level in the intestine. This suggests that the decrease in the blood level of serotonin was due to the inhibition of its synthesis in the brain, whereas the decrease in the duodenum level of serotonin was due to the compensatory release to blood while its synthetic rate remained unaltered. Thus, the developing brain before the blood-brain barrier formation was shown to secrete serotonin into blood.

[Compensatory reaction during degeneration of arcuate nucleus dopaminergic neurons in rats].

The study has been carried out to verify the authors' hypothesis that degeneration of dopaminergic (DA-ergic) neurons of the hypothalamic tuberoinfundibular system and concomitant development of hyperprolactinemia are accompanied by involvement of compensatory synthesis of dopamine (DA) by non-dopaminergic neurons expressing single complementary enzymes of synthesis of this neurotransmitter. Degeneration of DA-ergic neurons was produced by a stereotaxic injection into the brain lateral ventricles of 6-hydroxydopamine (6-OHDA) - a specific neurotoxin of DA-ergic neurons. 14 and 45 days after the toxin administration there were determined concentration of prolactine in peripheral blood by methods of immunoenzyme and radioimmunological analyses as well as the DA amount in the arcuate nucleus by the method of highly efficient liquid chromatography with electrochemical detection. In a part of the animals, slices were prepared from the mediobasal hypothalamus (arcuate nucleus and medial eminence) and perfused with Krebs-Ringer medium; then the DA concentration was determined in the slices and in the incubation medium. 14 days after the neurotoxin administration there were revealed an increase of blood prolactine concentration and a decrease of DA concentration in the arcuate nucleus in vivo as well a decrease of the total DA amount in the slices and incubation medium in experiments in vitro. 45 days after the neurotoxin administration, all the above parameters returned to the normal level. This, the obtained data indicate that the hyperlactinemia and DA deficit appearing during degeneration of the arcuate nucleus DA-ergic neurons seem to be compensated due to an enhancement of DA synthesis by non-dopaminergic monoenzyme neurons of arctuate nucleus.