E. V. Proshlyakova

Ontogenesis of the hypothalamic catecholaminergic system in rats: synthesis, uptake and release of catecholamines.

The development of the hypothalamic catecholaminergic system during ontogenesis in rats has been studied with glyoxylic acid histofluorescent method in vivo and with isotopic biochemical technique in vitro. It has been demonstrated that at the 15th fetal day the catecholaminergic system was functionally inactive at least in its ability for the uptake and K(+)-stimulated release of catecholamines. Since the 16th fetal day, hypothalamic neuronal elements gained an ability for synthesis of catecholamines, their specific uptake and K(+)-evoked release. Over the subsequent two days, the intensity of the fluorescent intraneuronal product rose considerably showing the increase of either synthesis or accumulation of catecholamines. Simultaneously, the values of the uptake and K(+)-stimulated release of the exogenous radioactively-labelled dopamine increased significantly. The intensity of the fluorescence of the hypothalamic neuronal elements dropped from 20th fetal until the ninth postnatal day, whereas the specific uptake doubled over the same period reaching its adult level. By the 21st postnatal day the reaccumulation of the fluorescent product occurred.

Development of the hypothalamic 5-hydroxytryptamine system during ontogenesis in rats: Uptake and release of 5-hydroxytryptaminein vitro

The development of the hypothalamic 5-hydroxytryptamine system has been evaluated in vitro according to [3H]5-hydroxytryptamine uptake and release in fetuses (16-20th fetal day), neonates (9th postnatal day) and adults (45th day of life). At the 16th fetal day the hypothalamic neural elements were characterized by specific uptake of 5-hydroxytryptamine and its spontaneous release; the next day K+-stimulated Ca2+-dependent release appeared. By the 18th fetal day, the 5-hydroxytryptamine uptake doubled and was retained at this level both in older fetuses and in postnatal rats. The K+-stimulated release of [3H]5-hydroxytryptamine increased considerably during the perinatal period, reaching an adult level by the 9th postnatal day. These data indicate the sprouting of 5-hydroxytryptamine fibers to the hypothalamus and the maturation of their membrane mechanisms for the bidirectional transport of 5-hydroxytryptamine early during ontogenesis-to a significant extent before the 18th fetal day.

Hypothalamo-pituitary control of the cell-mediated immunity in rat embryos: role of LHRH in regulation of lymphocyte proliferation.

Abstract The role of the neuroendocrine system in the development of cell-mediated immunity has been studied in fetal rats. The spontaneous and mitogen-induced proliferation of liver lymphocytes and thymocytes was evaluated in vitro in rats at the 22nd prenatal day following surgical ablation of the forebrain (encephalectomy) or of the entire brain and pituitary (decapitation) in rat fetuses in utero at the 18th day. Non-operated and sham-operated fetuses served as controls. The ablation of the entire brain and pituitary in rat fetuses resulted in an increase (40–60%) of spontaneous proliferation of liver and thymic cells in comparison with sham-operated fetuses. The ablation of the forebrain including the hypothalamus caused a decrease in the mitogenic proliferative response of thymocytes and liver lymphocytes for 40 and 20%, respectively. The ablation of the entire brain including the hypothalamus and pituitary resulted in a 80% decrease of the proliferative response of thymocytes and in the full suppression of proliferation of liver lymphocytes. The immune proliferative response was restored by the LHRH administration either systemically to operated fetuses (0.2 μg/fetus) or to the cell culture (10 −9 and 10 −7 M). It was concluded that the central nervous system was important for maturation of the immune system in rats during the prenatal period. In particular, neuroendocrine system are likely to play a major role as LHRH treatment in vitro and in vivo appeared to contribute to this regulation.

Pharmacological model of catecholamine depletion in the hypothalamus of fetal and neonatal rats and its application

Summary1.The present study aimed to develop a pharmacological model of catecholamine (CA) depletion in the hypothalamus during the period of its morphofunctional development, i.e. in fetal and neonatal rats of both sexes.2.In the first series of experiments, pregnant females and, hence, fetuses were systemically treated daily from the embryonic day (E) 13 to E20 with the inhibitor of the CA synthesis α-methyl-m-tyrosine. The CA concentrations were subsequently measured in the fetal hypothalamus at E21 by high performance liquid chromatography with electrochemical detection (HPLC-ED). In the second series of experiments, neonatal rats were injected with neurotoxin, 6-hydroxydopamine and/or α-methyl-m-tyrosine daily from the 2nd postnatal day (P2) to P10.3.The HPLC-ED assay of hypothalamic catecholamines (CAs) at E21 and P11 showed that both in fetuses and neonates, α-methyl-m-tyrosine caused more than 50% depletion of hypothalamic noradrenaline and adrenaline, while the dopamine (DA) level remained unchanged. The combined treatment of neonatal rats with α-methyl-m-tyrosine and 6-hydroxydopamine resulted additionally in a 25% decreased level of DA.4.The influence of CA deficiency on the developing hypothalamic CA system was further evaluated by measuring [3H]DA uptake by nervous tissuein vitro.5.The CA deficiency caused a 50% drop of [3H]DA uptake by the hypothalamic tissue in treated fetuses suggesting a stimulating effect of CAs on the early development of the CA system. In pharmacologically treated neonatal rats [3H]DA uptake remained at the control level showing no influence of the CA deficiency on the developing CA system after birth.6.The usefulness of the proposed pharmacological model for studying of CA influence on differentiating hypothalamic target neurons is discussed.

Hypothalamo–Pituitary System Controls the Development of Humoral Immune Response during Prenatal Ontogenesis in Rats

We studied the influence of the neuroendocrine system on the development of humoral immune response to sheep erythrocytes in rat fetuses. The removal of brain in utero by decapitation of 18-day fetuses induced a fourfold increase in the number of antibody-forming cells in the liver, as compared to the unoperated fetuses. After the removal of the forebrain, including hypothalamus (encephalectomy), the number of antibody-forming cells was comparable to that in unoperated fetuses. The observed increase in the number of antibody-forming cells in the liver was not due to a disturbed migration of precursors of B-lymphocytes in the spleen, since their content in the spleen was also four times that in the encephalectomized and unoperated fetuses. The increased number of antibody-forming cells in decapitated fetuses could be due to an enhanced proliferative activity of the lymphocytes in the liver of these fetuses. It has been proposed that humoral immunity is controlled by the hypothalamo–pituitary–adrenal system already during prenatal development; the adrenocorticotropic hormone and glucocorticoids appear to be involved in this regulation.

Development of the mesencephalic and diencephalic catecholamine systems in human fetuses: uptake and release of catecholamines in vitro

Development of catecholamine (CA) systems of the ventral mesencephalon and diencephalon were studied in human fetuses at age 6, 8, 10 and 12 weeks, evaluating the CA specific uptake and K(+)-stimulated release with the isotopic biochemical technique. In the mesencephalon, the [3H]dopamine (DA) uptake was detected as early as 6 weeks, suggesting the existence of either CA neurons or fibers. This was followed by gradual increase of the [3H]DA uptake up to 10 weeks and a subsequent fall at 12 weeks. In the diencephalon, the uptake was first observed at 8 weeks, followed by its decrease at 10 weeks and subsequent increase at 12 weeks. The dynamic uptake is considered as a manifestation of the continuous differentiation of CA neurons and sprouting of CA fibers. In contrast to uptake, no CA release was detected in response to membrane depolarization in the diencephalon and mesencephalon at any age studied, suggesting a timing dissociation between the onset of the CA uptake and K(+)-provoked release in the course of neuron differentiation in human fetuses.

The brain is one of the most important sources of dopamine in the systemic circulation in the perinatal period of ontogenesis in rats

This study was designed to test the authors’ hypothesis that dopamine passes from dopamine-synthesizing cells in the brain to the systemic circulation prior to the formation of the blood-brain barrier during ontogenesis. High-performance liquid chromatography studies demonstrated that peripheral blood dopamine levels before formation of the blood-brain barrier-in rat fetuses and neonates-are significantly higher than after formation of the barrier in adult rats, providing indirect evidence in support of the hypothesis. Furthermore, formation of the blood-brain barrier is accompanied by a significant increase in dopamine levels in the rat brain. Direct evidence for the hypothesis was obtained in the form of a sharp decrease in blood dopamine levels in fetuses after lesioning of dopamine-synthesizing neurons in the brain by encephalectomy.

The brain is one of the sources of L-dihydroxyphenylalanine in systemic circulation in fetuses and neonatal rats

The performed study was aimed at checking our hypothesis that the developing brain is a source of L-dihydroxyphenylalanine (L-DOPA), a precursor of dopamine in the total circulation system. At the initial stage, the L-DOPA concentration in peripheral blood was analyzed at the 18th and 21st embryonal days (E18 and E21), at the 3rd postnatal day (P3), and at the prepubertal period (P30). The highest L-DOPA concentration was revealed at the perinatal period, while decreased 4–12 times for the first month of life. The subsequent analysis of dynamics of the total blood L-DOPA content showed that maintenance of the constant L-DOPA concentration at the perinatal period on the background of a gradual increase of the blood serum volume is due to a rise of its secretion. At the postnatal period (P3–P30), the blood L-DOPA content increased twice in males, whereas it decreased to the same extent in females. Analysis of the L-DOPA concentration in two most important brain centers, hypothalamus and mesencephalon-rhombencephalon, showed its twofold decrease in hypothalamus during E18–E21 of development; then it slightly increased from E21 to P3 and fell 4–5 times by P30. In mesencephalon-rhombencephalon, the L-DOPA concentration was slightly reduced from E18 to E21 (only in females), while on P3 it returned to the E18 level and decreased 7–9 times by P30. The direct proof for the L-DOPA release from the developing brain into the systemic circulation follows from comparison of the blood L-DOPA concentration in shamoperated and encephalectomized rat fetuses after mechanical destruction of neurons of the two abovementioned most important dopaminergic centers. Thus, encephalectomy led to a twofold reduction of the blood L-DOPA concentration (statistically significant differences were observed only in females). Thus, the work presents evidence that the developing brain is one of L-DOPA sources in the total circulation system in rats during prenatal and early postnatal periods of ontogenesis.

FUNCTIONAL ACTIVITY OF CATECHOLAMINERGIC SYSTEM IN HUMAN FETAL MIDBRAIN AND DIENCEPHALON

The development of catecholaminergic system of the midbrain and diencephalon was studied in human embryos and fetuses aged 6, 8, 10, and 12 weeks by specific capture and K+-stimulated release of3H-dopaminein vitro. Specific capture of3H-dopamine was first detected in the midbrain of 6-week embryos and in the diencephalon of 8-week fetuses. The time course of the capture points to on-going differentiation of catecholaminergic neurons and fiber growth and the presence of the caudorostral gradient in the development of brain catecholaminergic system. The release of catecholamines was not stimulated in response to membrane depolarization in the midbrain and diencephalon at any of the studied stages of development. The difference in the time of capture and K+-stimulated release of catecholamines is related to specific features of differentiation of these neurons in human fetuses.

Development of the mesencephalic and diencephalic serotoninergic system in mice and the role of serotonin in this process

The development of functional activity of the serotoninergic system in the mesencephalon and diencephalon of mice is followed during ontogeny, and it is found that serotoninergic neural elements become capable of specific serotonin uptake and K+-stimulated serotonin release on day 17 of prenatal life. A single serotonin injection into a female mouse on day 8 of gestation resulted in a drastically reduced specific3H-serotonin uptake by the brain of its 18-day fetuses.