Anna Sapronova

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.

Dopamine turnover in the mediobasal hypothalamus in rat fetuses

In this study, the dopamine turnover in the mediobasal hypothalamus, the key compartment of the neuroendocrine regulation of reproduction, was evaluated in fetal male and female rats. High-performance liquid chromatography with electrochemical detection was used to measure 3,4-dihydroxyphenylalanine, dopamine and 3,4-dihydroxyphenylacetic acid in the mediobasal hypothalamus of fetuses on the 21st day of intrauterine development and in primary cell culture (cell extracts and culture medium) of the same brain region, explanted at the 17th fetal day and maintained for seven days. The same technique was applied to determine dopamine release from fetal neurons of the mediobasal hypothalamus in response to an excess of K+ in the perifusion system or in culture. L-3,4-Dihydroxyphenylalanine, dopamine and 3,4-dihydroxyphenylacetic acid were detected both ex vivo and in culture. The ratios of the concentrations of L-3,4-dihydroxyphenylalanine/dopamine and 3,4-dihydroxyphenylacetic acid/dopamine were significantly higher in vitro than ex vivo, showing a lower rate of dopamine production and a higher rate of its degradation in the experiments in vitro. Moreover, it has been demonstrated that an excess of K+, i.e. a membrane depolarization, resulted in a highly increased release of dopamine in the perifusion system and in culture. The dopaminergic activity in the developing mediobasal hypothalamus showed sexual dimorphism that was manifested in a greater concentration of 3,4-dihydroxyphenylalanine and dopamine, at least in cell extracts of cultures, as well as in a higher rate of dopamine release, both in the perifusion system and in culture in males compared to females. Thus, dopamine is synthesized and released in response to a membrane depolarization in the mediobasal hypothalamus of rats as early as the end of intrauterine development, suggesting its contribution to the inhibitory control of pituitary prolactin secretion.

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.

Developing brain as an endocrine organ: Secretion of dopamine

This study was aimed to test our hypothesis that the developing brain operates as an endocrine organ before the establishment of the blood-brain barrier (BBB), in rats up to the first postnatal week. Dopamine (DA) was selected as a marker of the brain endocrine activity. The hypothesis was supported by the observations in rats of: (i) the physiological concentration of DA in peripheral blood of fetuses and neonates, before the BBB establishment, and its drop by prepubertal period, after the BBB development; (ii) a drop of the DA concentration in the brain for 54% and in blood for 74% on the 3rd postnatal day after the intraventricular administration of 50 μg of α-methyl-p-tyrosine, an inhibitor of DA synthesis, with no changes in the DA metabolism in peripheral DA-producing organs. Thus, the developing brain is a principal source of circulating DA which is capable of providing an endocrine regulation of peripheral organs and the brain.

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.

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.