Ca2+-dependent secretion of serotonin by the rat hypothalamus during ontogenesis

A study was made of functional maturity of the terminal part of serotoninergic system of rat hypothalamus in perinatal period: the maturity was estimated by the ability to release serotonin. The release of specifically taken up serotonin (3H-5-OT) by the tissue of hypothalamus of 16-20-day-old rat fetuses, 8-9-day males and adult males was studied in the perfusion system. Spontaneous release of the labelled amine was recorded in the earliest studied period--on the 16th day of the prenatal period, but the response to K+ depolarization was absent at this time. For the first time the increase of the rate of 3H-5-OT release in response to depolarization was noted on the 17th day of development. In the absence of Ca2+ depolarizing stimulus produced no increase in the release of the labelled product. Similar results were obtained with perfusion of fetal hypothalamus on the 18t hand 20th days of development. In neonatal animals the release of 5-OT in response to depolarization was equal to that in adults. The data obtained point to a possible functioning of serotoninergic elements of hypothalamus in the perinatal period in rats.     

Effects of male sex hormones on specific uptake and release of 3H-serotonin in the rat hypothalamus in vitro]

With the use of "isotopic method" a study was made of the main parameters of functional activity of serotoninergic elements of hypothalamus--the specific uptake and release of 5-OT. The animals used were sexually mature rats castrated on the first postnatal day. In sexually mature intact males the specific uptake of 3H-5-OT by serotoninergic structures of the anterior hypothalamus was significantly lower than in females. Castration of animals on the first day of life resulted in the increase of specific 5-OT uptake in sexually mature males up to that observed in females. There were no differences between the sexes in the rate of spontaneous release of 5-OT. However, response to K(+)-depolarization in the anterior hypothalamus of intact males was significantly lower than that in females. In the hypothalamus of males castrated neonatally the amplitude of the response to the effect of the depolarizing agent was increase up to the level observed in females. By the results obtained it is indicated that elimination of the effect of male hormones on the first postnatal day results in the increase of 5-OT uptake and release in the hypothalamus of sexually mature rat males.     

Radioautographic study of the hypothalamic serotoninergic structures in the perinatal period of the rat

The distribution of 3H-serotonin-binding structures in hypothalamus of 16 and 18 day old fetuses and of 9 day old rats was studied after intraventricular injection of 3H-serotonin. Rare 3H-serotonin-binding little differentiated cells were found predominantly in the intermediate zone of the 3rd ventricle in the retrochiasmatic area wall on the 16th and 18th days of prenatal development. In addition, an aggregate of heavily labeled neurones was observed in the suprachiasmatic area. At the same time 3H-serotonin-binding fibers first appeared, predominantly in the optic chiasma and perichiasmatic area. Radioactively labelled cells, which can be characterized by their morphology as differentiated neurones, were located in the dorsomedial nucleus on the 9th day of postnatal development. The number of serotonin-binding fibers markedly increased but the pattern of their distribution was, on the whole, similar to that in fetuses. The data obtained suggest that the main stages of structural organization of serotoninergic system of hypothalamus in rats are realized during perinatal period.     

Further characterization of the process of in vitro uptake of radiolabeled copper by the rat brain

We have previously demonstrated that hypothalmic slices obtained from adult male rats accumulate 67Cu by two ligand-dependent, saturable processes: a high and low affinity process. To further establish the generality of these uptake processes, we defined the ligand requirements and the saturation kinetics of 67Cu uptake by tissue slices obtained from the newborn hypothalamus (HT); adult male hypothalamus, hippocampus, cortex, median eminence, and caudate nucleus; hypothalamus and hippocampus of castrated (14 days) males and of pregnant (19 days) and ovariectomized (14 days) females. It was found that ionic 67Cu2+ was poorly taken up by newborn HT and adult caudate, complexation with His enhanced 67Cu uptake 3-4-fold, and complexation with albumin inhibited 67Cu uptake. These ligand requirements are identical to those we have previously shown for the adult HT. When 67Cu uptake was evaluated under conditions optimal for the high or the low affinity process, for each process the dose response curves generated from these various tissues were very similar. In addition, we assessed the uptake of both components of the CuHis2 complex by incubating tissues with 67Cu3 H-His2 and found that the tissue ratio of 67Cu:3H was a sigmoidal function of the concentration of the Cu complex such that at greater than 5 microM, the ratio was about 3-fold greater than the medium ratio; indicating preferential uptake of 67Cu relative to 3H-His. The changes in isotope ratios were observed in newborn HT and adult HT, as well as caudate. These similarities in the ligand requirements and saturation kinetics of 67Cu uptake establish the generality of these two processes of in vitro uptake of copper in the rat brain.     

Spleen cells from adult mice given total lymphoid irradiation (TLI) or from newborn mice have similar regulatory effects in the mixed leukocyte reaction (MLR). II. Generation of antigen-specific suppressor cells in the MLR after the addition of spleen cells from newborn mice

Spleen cells from newborn BALB/c mice were added to the mixed leukocyte reaction (MLR) between a variety of responder and stimulator cells. The newborn cells nonspecifically suppressed the uptake of (3H)-thymidine and the generation of cytolytic cells regardless of the responder-stimulator combination used. Suppressor cell activity fell rapidly during the first 4 days after birth, and could not be detected by day 20. Newborn spleen cells inhibited the generation of nonspecific suppressor cells during the MLR but did not inhibit the generation of antigen-specific suppressor cells. Thus, newborn spleen cells exhibit a pattern of regulation of the MLR similar to that reported previously for spleen cells from adult mice given total lymphoid irradiation (TLI). These regulatory interactions provide a model that explains the ease of induction of transplantation tolerance in vivo in newborn mice and in TLI-treated adult mice.     

The significance of a positive reaction to anti-serotonin (5-HT) in certain hypothalamic neurons of the rat during the perinatal period

In this study, two regions containing 5-HT immunopositive neurons were observed in the hypothalamus of fetal (18th fetal day) and neonatal rats (9th postnatal day) after pretreatment with the monoamine oxidase inhibitor, pargyline, and the amino acid precursor of the 5-HT synthesis, l-tryptophan. They were localized in the anterolateral hypothalamus and the dorso-medial hypothalamic nucleus. Immunostaining was completely blocked by the preliminary injection of fluoxetine, a specific uptake inhibitor of the serotoninergic neurons. Thus, the 5-HT immunostaining of hypothalamic neurons reported in this study is accounted for by the specific uptake of the 5-HT from the environment rather than by its synthesis from l-tryptophan.     

Ontogeny of the pig hypothalamic neurosecretory system with particular reference to the distribution of neurophysin

The immunoperoxidase cytochemical reaction was applied to the localization of neurophysin-containing elements in the fetal and adult pig hypothalamus. In the 60 day fetal pig, cells of the supraoptic nucleus (SON) were the only structues in the hypothalamus in which neurophysin was detected. However, by 87 days the cell bodies in both the SON and paraventricular nucleus (PVN) contained neurophysin-like material. The distribution of immunoreactive material in the 111 day fetal animal was similar to that found in the adult pig. In transverse section of the mature pig the SON exists in two discrete components; an antero-lateral group of cells connected by scattered cells to a smaller postero-medial group. Anteriorly, the PVN appears as a line of cells bordering the third ventricle but as we proceed posteriorly the dorsal aspect expands laterally to give a wedge-shaped group of cells. In mid-sagittal sections, the cells of the PVN are distributed over a wide area of the anterior hypothalamus in a triangular profile. The borders between the SON and PVN became more difficult to define in medial sections than in lateral sections. Continuous gradient polyacrylamide gel electrophoresis was carried out on the neural lobe extracts from fetal, newborn and adult pigs. Proteins with an electrophoretic mobility similar to that of porcine neurophysins-I, -II and -III were present in the newborn and 98 day fetal pig. It is concluded that material immunoreactive with anti-neurophysin serum is present in the hypothalamus of the 60 day fetal pig. Furthermore, at late fetal development and during the postnatal period it is tentatively suggested that the neurophysin present in the pituitaries of these animals is chemically identical with that of adult neurophysin.     

Immunocytochemical and autoradiographic research on the growth of serotoninergic fibers to the cerebral ventricles in the perinatal period in rats

Anatomical relationships between serotoninergic (5-HT) fibers and cerebral ventricles were studied in rats from the 16th fetal day until the 9th postnatal day with immunocytochemistry and radioautography. In the latter case, 5-HT neuronal elements were detected according to their specific uptake of intraventricularly injected 3H-5-HT. On the 16th fetal day, occasional 5-HT fibers first spread from the main place of their origin in the raphe nuclei to the dorsocaudal portion of the 3rd ventricle and aqueduct. Two days later, a more extensive network of 5-HT fibers appeared around the dorsal portion of the 3rd ventricle, whereas fibers only rarely penetrated fibers became noticeable in the lateral and 3rd ventricles. The functional significance of hypothalamic and ventricular 5-HT is discussed from the standpoint of its being either a modulator of growth and differentiation of the developing brain, or a factor involved in some specific neuroendocrine functions.     

Changes in the intracellular accumulation and distribution of metallothionein in rat liver and kidney during postnatal development

Metallothionein (MT) bound to zinc and copper was detected in high concentration in fetal and newborn rat livers by a cadmium saturation method. The levels of both hepatic zinc and MT remained high for the first 14 days after birth and decreased to adult levels by 24 days of age. There was a direct linear relationship between hepatic metallothionein and zinc concentrations during the first 31 days after birth. The ratio of MT to zinc levels also decreased with age suggesting a rapid degradation of MT during postnatal development. Immunohistochemical localization of MT by peroxidase-antiperoxidase technique, using a specific antibody to MT, showed intense intranuclear staining for MT in fetal and newborn rat liver which persisted until Day 9. The nuclear MT staining decreased with age; at 11 days it was equal both in nucleus and cytoplasm and at 14 days, MT was localized mainly in the cytoplasm, similar to adult rat liver pattern. The intranuclear localization of MT in neonates could be considered as a typical fetal-neonatal morphological pattern and its subsequent presence in the cytoplasm, an adult pattern.     

Serotonin contents in the hypothalamus of adult males, females and males castrated during the neonatal period

The aim of the present work was to study the character of the change in serotonin level in the anterior and medial basal hypothalamus of adult rats after the effect of testicular hormones had been switched off on the first day of postnatal life. It was shown in our work that in males serotonin level was significantly lower than that in females by 67 and 46% in the anterior and medial basal hypothalamus, respectively. Castration of newborn males resulted in a significant increase in serotonin level in both anterior and medial basal hypothalamus-up to the level observed in females. It is supposed that the male sex hormones affect differentiation of serotoninergic system of the brain.     

Morphofunctional characteristics of serotonin-like neurons in the hypothalamus of rats in ontogeny

An attempt has been made to reveal 5-HT immunopositive (IP) neurones in the hypothalamus of intact foetuses (18th day of gestation) and neonatal (9-day) rats under normal conditions and after their treatment with drugs involved into 5-HT metabolism or into regulation of its uptake by serotoninergic neurones. 5-HTIP cells were not observed in intact animals as well as after L-tryptophan treatment, whereas two large colonies of these neurones were found in the anterio-lateral hypothalamus and dorsomedial nucleus after subsequent injections of monoamine oxidase inhibitor, pargyline, and amino acid precursor of 5-HT synthesis, L-tryptophan. Significantly less intensive reaction was observed after injections of another precursor of 5-HT synthesis, 5-hydroxytryptophan, or pargyline only. Immunostaining evoked by pargyline or L-tryptophan can be prevented by preliminary injections of fluoxetine, a specific inhibitor of 5-HT uptake by serotoninergic neurones. These data suggest that the immunostaining of hypothalamic neurones is due to their capacity to take up specifically 5-HT from the environment rather than to its intraneuronal synthesis from L-tryptophan. However, 5-HT synthesis from 5-hydroxytryptophan in the same cells may also take place. The uptake of extracellular 5-HT by catecholaminergic neurones is absent, since nomifensine, a specific inhibitor of this uptake, does not affect immunostaining.     

Postnatal changes of cathepsin D activity in rat liver and brain

Total and specific activity of cathepsin D (EC. 3.4.23.5) were measured in rat liver and brain from 1 to 98 days of age. The activity of cathepsin D in the liver of adult and newborn rats was the same while in the rat brain it was higher in adult than in newborn rats. In the liver maximum specific activity of cathepsin D occurred on the 10th postnatal day and minimum on the fourth day of age. In the brain maximum specific activity of the enzyme occurred on the 14th postnatal day. Total activity of cathepsin D increased after birth in rat liver and brain. These results are discussed in relation to the functional role of cathepsin D in the rat liver and the brain.     

Are 5-HT receptors involved in the sprouting of serotoninergic terminals following neonatal 5,7-dihydroxytryptamine treatment in the rat?

The neonatal administration of 5,7-dihydroxytryptamine to rats (100 mg kg^?1 s.c. on the 1st and 2nd day after birth) resulted in marked reductions in serotoninergic presynaptic markers ([³H]-5-HT synaptosomal uptake, tryptophan hydroxylase activity and endogenous 5-HT content) in various forebrain areas, particularly the cerebral cortex and the hippocampus. In contrast, this treatment produced an increased outgrowth of serotoninergic terminals in the brain stem as judged by the significant increments of these presynaptic markers in this region. Both in the hippocampus and the brain stem, these 5,7-dihydroxytryptamine-induced changes in serotoninergic innervation were associated with a transient increase in 5-HT-sensitive adenylate cyclase activity. No significant alteration of the specific high affinity binding of [³H]-5-HT to synaptosomal membranes from various brain regions was detected in 5,7-dihydroxytryptamine-treated rats for at least the first postnatal month.The chronic blockade of 5-HT receptors by metergoline (5 mg kg^?1 day^?1 from day 3 to day 22 after birth) altered neither the changes in presynaptic markers nor the evolution of [³H]-5-HT high affinity binding in 5,7-dihydroxytryptamine-treated rats.These findings further illustrate that the high affinity binding sites for [³H]-5-HT do not correspond to postsynaptic 5-HT receptors coupled to adenylate cyclase in the rat brain. Apparently, 5-HT receptors play no role in the increased outgrowth of serotoninergic systems in the brain stem following neonatal 5,7-dihydroxy-tryptamine treatment.     

The effect of constant light and chemical sympathectomy on the development of serotonin, n-acetyltransferase and monoamine oxidase activities in the rat epiphyses.

The daytime activity of N-acetyltransferase per mg epiphysis decreased to 1/10 the newborn level by the age of 13 days, and subsequently remained unchanged. The night activity was equal to the day activity at the age of 3 days, was higher at the age of 6 days and was 20 X that of the day level at the age of 40 days. Keeping animals in constant light after birth depressed the development of these diurnal differences in N-acetyltransferase activity and slowed down the decrease in enzyme activity after birth. Sympathectomy with 6-hydroxydopamine after birth abolished the development of the diurnal rhythm in N-acetyltransferase in 12-day-old rats in two experiments out of five and only decreased the night activity without abolishing the rhythm in three experiments. Monoamine oxidase activity (MAO) per mg epiphysis, which is the same in newly born and in adult animals, decreased to half the original value for 6 days after birth and then increased again. Constant light after birth did not influence MAO activity, but sympathectomy with 6-hydroxydopamine decreased activity in the epiphysis in 12-day-old animals.     

Detection of antigens of endogenous viruses of the C-type during mouse development]

Embryos of the 12th-20th day of gestation, newborn and adult AKR and BALB/c mice were investigated for the presence of mouse C-type virus major structural p30 protein (gs-1) and Gross leukemia virus type-specific antigen AGLV) by means of radioimmunodiffusion with test systems. The p30 protein was distinctly determined from the 12th day of intrauterine development in both mouse lines; it persisted in the embryo tissues until birth and was detectable also in the AKR and BALB/c mouse tissues from the first days of postnatal development and during the whole life. AGLV was not revealed in BALB/c and AKR embryos and in adult BALB/c mice; however it was found in the AKR newborn mice since the 1st-2nd day after birth. Basing on these data a conclusion was drawn that p30 protein and AGLV were expressed independently according to the radioimmuno-diffusion method sensitivity.     

Development of serum lipoproteins from fetal to adult rats]

1. The semi-quantitative development of lipoproteins in the serum (beta-, pre-beta- and alpha-lipoproteins) from fetal (day 18 of intra-uterine development) to adult age was determined by agarose gel electrophoresis and determination of total lipids. 2. A high proportion of beta- and a very low proportion of pre-beta-lipoprotein is typical of the fetal stage. In adult age, however, the beta-lipoproteins are the smallest fraction, and the pre-beta-lipoproteins, the second-strongest fraction. 3. The transition from fetal to adult lipoprotein pattern is more or less abrupt, with the change of the proportion for each of the three lipoprotein fractions occurring at different intervals from birth. Adaptation of the fetal lipoprotein status to that of the adult animal in regard to quantity and mutual relationship of the fractions was observable, at the earliest, 7 weeks after birth.     

Newborn Rabbit Blood–Brain Barrier Is Selectively Permeable and Differs Substantially from the Adult

Abstract: Examination of blood-brain barrier (BBB) function by the intracarotid injection technique has been utilized in studies of newborn (6–30 h) and adult rabbits. The exclusion of mannitol (mol. wt. 182), dextran (mol. wt. 60,000–90,000) and indium-bound EDTA indicate that the newborn BBB has restrictive properties similar to the adult. At birth, saturable, carrier-mediated transport mechanisms are present, regulating the entry of glucose, amino acids, organic acids, purines, nucleosides and choline. No difference in brain uptake of glucose was observed between adult and newborn, but considerably higher uptake rates for arginine, choline and adenine were seen in the newborn. In contrast to suggestions of an immature barrier in young animals, these studies indicate that a sophisticated, selective BBB is operative at birth. Furthermore, the specific selectivity and dramatic increases seen for certain metabolites imply a vital function in the newborn for these carrier systems.     

Premature induction of amylase in pancreas and parotid gland of growing rats by dexamethasone.

Studies were made on changes in the contents of alpha-amylase (EC 3.2.1.1) in the pancreas and parotid gland of rats during postnatal development, on the premature induction of this enzyme by hormones and on the existence of specific glucocorticoid receptors in these tissues. The amylase content in the pancreas increased from the 9th day after birth and reached the adult level on the 28th day, its content in the parotid gland increased rapidly from the 16th to the 28th day after birth and then rose more gradually to the adult level. Injection of dexamethasone into rats 6--8 days after birth induced increase in the amylase of the pancreas but not the parotid gland. However, injection of dexamethasone into weanling rats 21--23 days after birth resulted in precocious induction of amylase in both tissues. Specific glucocorticoid receptors were detectable in the parotid gland of rats from 6 days after birth but were almost undetectable in the pancreas until adolescence.     

Ontogeny of pituitary responsiveness to corticotropin-releasing hormone in rat

The ontogeny of the pituitary's responsiveness to synthetic rat corticotropin-releasing hormone (CRH) in the late prenatal and early postnatal periods of rats was studied by a superfusion system using whole pituitaries. A significant increase of immunoreactive beta-endorphin (IR-beta-Ep) secretion in response to 10(-10) M CRH but not to 10(-11) M CRH was observed in pituitaries from the 15th day of gestation, the earliest day that we tested, whereas 10(-11) M CRH stimulated IR-beta-Ep release from the pituitaries of 17.5-day-old fetuses. Dose-related IR-beta-Ep secretions induced by 10(-12) M to 10(-10) M CRH were observed in pituitaries of 19.5- and 21.5-day-old fetuses, and 1-, 3- and 9-day-old newborn pups. CRH stimulated not only IR-beta-Ep and IR-adrenocorticotropic hormone (ACTH) but also IR-alpha-melanocyte-stimulating hormone (IR-alpha-MSH) secretions from fetal pituitaries. The content of IR-CRH in the hypothalamic extract from 15-day-old fetus was 6.6 +/- 3.6 pg/hypothalamus (mean +/- S.E.M.) and it gradually increased to reach 212.7 +/- 20.3 pg/hypothalamus on the 21.5th day of gestation. However, the content of IR-CRH in the hypothalamus dramatically decreased just after birth and then rapidly increased again from the 5th day after birth. These data indicate that the responsiveness of corticotrophs to CRH is already present on the 15th day of gestation, when the content of IR-CRH in the hypothalamus is extremely low and that the amount of hypothalamic IR-CRH dramatically dropped for several days just after birth in rats.     

An immunohistochemical analysis of rat hippocampal antigens in early postnatal ontogeny by using monoclonal antibodies]

In order to study the molecular mechanisms of neurogenesis, monoclonal antibodies (MAbs) were produced against antigens of the developing rat hippocampus. MAb 3G7-F8 was used for immunohistochemical localization of the corresponding antigen of paraffin sections of the rat brain at days 0, 5, 14, and 21 of the postnatal development. In the hippocampus of newborn and 5-day-old rats, positive immunostaining was observed in the cytoplasm and proximal segments of processes of neurons located in granular, polymorph, and pyramidal layers, as well as in entorhinal cortex. In granule cell bodies and neurons of entorhinal cortex specific staining decreased by day 14 and disappeared by day 21 after birth, whereas neurons of pyramidal and polymorph layers remained immunopositive. Diffuse specific staining in the cerebellum was observed beginning from day 5 after birth in the Purkinje cell layer. On days 14-21 positive reaction was observed in Purkinje cell bodies and in the layer containing dendrites of Purkinje cells and parallel fibers. External and internal granular layers remained immunonegative. No specific staining was observed in other regions of the brain, as well as in the control slices. These data suggest that the antigen detected by the 3G7-F8 antibody is involved in the formation of the neuronal connections.