The uptake of 3H-serotonin by neural elements of the rat hypothalamus in ontogeny

Development of serotoninergic system of rat hypothalamus has been studied. The level of its maturity was measured by specific absorption of 3H-serotonin by hypothalamus of fetuses (days 16-20 of intra-uterine development), newborn (9 days after birth) and adult rats. Inhibitors of specific serotonin capture by serotoninergic neural elements (cocaine, fluoxetin and cytalopram) were used in control experiments. Specific uptake of 3H-serotonin by hypothalamus was detected on day 16 of intra-uterine development. It increased twice by day 18 and remained unchanged until birth. No statistically significant increase of 3H-serotonin uptake was detected in newborn and adult animals.     

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.     

Nerve fiber interrelationships with the ependyma of the 3d ventricle in rats in the perinatal period]

The localization and ultrastructure of nerve fibers in the wall and lumen of ventral region of III ventricle was studied from the 16th day of prenatal period till the 9th day of postnatal period in the Wistar rats. The nerve fibers were first found in the subependyme zone; between the ependyme cells and in the lumen of ventricle on the 18th day of development. The nerve fibers occur constantly in the lumen of ventricle beginning from the 3rd day of postnatal period. The dilatations of nerve fibers contain granules of 50 to 90 nm in diameter with the electron-dense center (carriers of monoamines and/or releasing hormonnes) granules with moderately osmiophilic contents and light vesicles of the same size. It is suggested that these structures reflect different stages of the release of neurohormones in the cerebrospinal fluid. The synapse-like contacts of nerve fibers with the ependyme cells are observed beginning from the 3rd day of postnatal period. Their role in the regulation of absorption of substances by the ependyme cells functioning is discussed.     

The formation of catecholaminergic structures in the mediobasal hypothalamus of the rat

A study was made of the formation of catecholaminergic system in the medial basal hypothalamus of Wistar rat fetuses using histofluorescent method modified by de la Torre. In the periventricular region of 16-day fetuses the cells with green fluorescence characteristic for catecholamines were found. In 18-day fetuses catecholaminergic cells were found in paraventricular, arcuate and dorsomedial hypothalamic nuclei. In all the nuclei studied the number of catecholaminergic fibers and terminals of various size is greatly increased. The data obtained suggest that medial basal hypothalamus of 18-day fetuses has a complex catecholaminergic system.     

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.     

Evidence for the effectiveness of the blood--CSF barrier in the fetal rat choroid plexus. A freeze-fracture and peroxidase diffusion study

The blood--cerebrospinal fluid (CSF) barrier in the choroid plexus is principally constituted of apical junctional complexes between epithelial cells. The effectiveness of this barrier was studied during the fetal development in the rat. Choroid plexuses from fetuses (14th and 18th embryonic day) and newborn (1 and 6 day old) rats were examined after intravascular administration of a proteic tracer (horseradish peroxidase) and investigated by freeze-fracture. From the 14th day of fetal life, apical junctions were seen to constitute a barrier that prevents the passage of peroxidase from blood to CSF; the tight junctions were morphologically similar to those of the mature animals; the junctional fibrils appeared continuous on complementary replicas. These data suggest that, from the 14th day of fetal development, the blood--CSF barrier is both morphologically and physiologically mature.     

In vivo and in vitro studies on the appearance of LHRH neurons in the hypothalamus of perinatal rats

Summary Ontogenetic development of LHRH-containing neurons was studied by fluorescence and enzyme immunohistochemistry in rats. In in vitro studies, the tissues of the septal-chiasmatic and mediobasal hypothalamic areas of fetal rats on day 16.5 or 18.5 of gestation were trypsinized separately for dissociation of the neural cells, and cultured for several days. Immunopositive reaction against LHRH was first detected in nerve cells derived from both areas of the hypothalamus of the fetuses on days 16.5 and 18.5 of gestation, after 8 and 6 days culture, respectively. The cells were small, and seemed to be bipolar in morphology indicating an axon and arborized dendrites. Immunopositive material occurred in the cell soma as well as in the cellular processes. In in vivo studies, immunopositive material, possibly deposited in nerve fibers, appeared first in OVLT and simultaneously in the external layer of the median eminence of fetuses on day 20.5 of gestation. The immunoreactive fibers increased in number in both parts with development, especially after birth in the median eminence. No immunopositive material was detected within any neural cell bodies nor in the cytoplasm of any ependymal cells.This work was financed by the Ministry of Education, Japan. No. 257008. We would like to thank Dr. Katsuhiko Saito (Department of Surgery, Tokushima University) for his kind advice on the preparation of the antibody used for the immunofluorescence study.     

Ultrastructural study on the hypothalamic-hypophysial-adrenal axis in fetal rats

Summary The adrenal glands of decapitated and encephalectomized fetal rats were investigated electron microscopically and compared to those of normal intact fetal rats. Although the adrenal cortices did not show three zones (zona glomerulosa, fasciculata, and reticularis) on the 16.5th day of gestation when the decapitation or encephalectomy was carried out in utero, the zonation was recognized in fetuses operated on the 21.5th day of gestation. The same was true for normal control fetuses. However, cytoplasmic characteristics suggesting steroidogenesis in the cortical cells were reduced to various degrees in the encephalectomized or decapitated fetuses, especially in the latter ones. The change in cytoplasmic appearance was more conspicuous in the inner portion of the cortex. This result suggests that for the maintenance of normal adrenocortical function the hypothalamus may be indispensable even during the prenatal life of rats.     

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.     

Hypothalamic influence on differentiation of the immunoreactive ACTH beta-LPH, alpha- and beta-endorphin containing cells in adenohypophysial primordia of rat fetus in organ culture (author's transl)]

Adenohypophysial primordia were isolated in rat fetuses from day 12.5 to day 15.5 of gestation. The organ culture employed for maintenance of the primordia was made up according to Watanabe et al. (1973). The fixation of primordia in Bouin Hollande's solution was performed after 9, 8, 7 or 6 days of culture when the normal duration of pregnancy was achieved. The cultivated primordia were immunologically studied using different antisera: anti-alpha(17-39)ACTH, anti-beta(1-24)ACTH, anti-beta-LPH, anti-alpha and anti-beta-endorphins, with immunoperoxidase or immunofluorescence techniques, including control experiments of the specificity of the antisera. A similar study was performed on pituitaries removed from normal rat fetuses from day 16.5 of gestation and each day up to birth, and fixated immediately. In vivo the first cells reacting with all the antisera used in this study were observed on day 16.5 of gestation; their number increased during gestation (Fig. 1 A, B and C). Immunoreactive cells with the different antisera could be detected in primordia isolated on day 12.5 of gestation after 9 days of culture. Numerous groups of cells were observed in primordia of older fetuses (Fig. 2 A and B). These data indicate that the corticotropic cells in rat fetuses could start to be differentiated without stimuli from the hypothalamus since primordia were isolated before the appearance of this cell type in normal rat fetuses and before the differentiation of the hypothalamus. The presence of ACTH and other peptides such as beta-LPH or beta-endorphin would support the hypothesis of a common precursor in this cell type existing early in gestation. Similar results were obtained in human fetuses.     

Light and electron microscopical investigations on the tanycyte differentiation during the perinatal period in the rat

Summary The differentiation of tanycytes was studied light and electron microscopically during the perinatal period in rats, the time when functional connections between hypothalamus and hypophysis are established. The 3rd ventricle is slit-like between 16 and 18 days of the prenatal period. Its wall is formed by intensively proliferating matrix cells with apical processes, ovoid perikarya and a basal process. The ventral region of the 3rd ventricle becomes funnel-shaped on the 20th day of the prenatal period. As the cells differentiate, the apical process becomes shorter and broader. Moreover, on day 20 of prenatal life cells without apical processes appear. Their number increases during the postnatal period. The concentration of endoplasmic reticulum, mitochondria, polysomes, lipid droplets, dense bodies (lysosomes), lamellated and multivesicular bodies increases. Initially the cells are similar but from the 3rd day of postnatal life differentiation occurs in different regions of the infundibular recess. After the 5th day, there are no marked changes in the structure and distribution of these cells.K. Chandrasekhar wishes to thank the Indian National Science Academy and USSR Academy of Sciences for the award of a fellowship during the tenure of which this work was completed     

Impaired spreading of surfactant phospholipids in the lungs of newborn rats with pulmonary hypoplasia as a model of congenital diaphragmatic hernia induced by nitrofen

In order to clarify the pathological outcome of congenital diaphragmatic hernia (CDH), we devised an animal model of CDH by administration of 2,4-dichlorophenyl-p-nitrophenyl ether (nitrofen) to pregnant rats, and determined the level and distribution of lung surfactant using the monoclonal antibody toward sphingomyelin and disaturated phosphatidylcholine (disat-PC). In control rats, the concentration of disat-PC was found to increase greatly from 16 to 18 days of gestation. Intragastric administration of nitrofen to pregnant rats at day 9 of gestation resulted in CDH in 42.7% of fetuses delivered after 20 days of gestation. In nitrofen-treated fetuses, the concentration of disat-PC in the lungs was lower than those in control fetuses, and surfactant apoprotein SP-A was similarly reduced in nitrofen-treated fetuses. However, the concentration of disat-PC in nitrofen-treated fetuses was higher than that in control fetuses at 18 days of gestation, indicating a synthetic potential of surfactant in nitrofen-treated fetuses comparable to that at the late stage of normal gestation. Immunohistochemical study with the antibody revealed that surfactant phospholipid was mainly in the form of intracellular granules in nitrofen-treated fetuses, probably causing the hypoplastic lungs and then CDH, in contrast to the uniform distribution on the pulmonary alveolar surface in control fetuses.     

Proliferation of piriform neurons in the rat cerebellar cortex in the pre- and postnatal periods of development]

The method of histoautoradiography with the use of H3-thymidine was applied to the study; there were established the periods of appearance in the cerebellar anlage of albino rats of neuroblasts differentiated into the piriform neurones of the cerebellar cortex; dynamics of the proliferative activity of these cellular elements in the course of the pre- and postnatal periods of development of the experimental animals was investigated. On the basis of the material obtained a conclusion was drawn that the last cell divisions (the result of cell differentiation were Purkinje's cells of the cerebellar cortex) stopped by the 13th--15th day of the embryonic development. No incorporation of the labeled precursor into the DNA of the nuclei of the differentiating piriform neurons occurred later.     

The prenatal morphogenesis of the lateral nasal wall in the rat (Mus rattus)

In the course of this investigation 24 fetuses of the Sprague-Dawley rats were used. Pregnant animals were sacrificed at 12, 14, 16, 18, and 20 days of gestation. The fetuses were removed and fixed in buffered formalin solution. The heads were dehydrated, embedded in paraffin, and the nasal cavities were sectioned serially. Light microscope observations were made on 10 to 15 μ thick sections stained with H and E, Mallory's trichrome stain, and Gomori's alkaline phosphatase stain. The 12 day old fetus possessed well developed olfactory depression with a thick epithelial covering. The olfactory fossa was still separated from the buccal cavity by a solid layer of young mesenchyme. In the 14 day old fetus the communication between the nasal and buccal cavities was established. The palatal shelves developed still vertically. All the turbinates and the nasal capsule were already differentiated. The glandular system was represented with the main and two to three accessory excretory ducts. During the sixteenth day of prenatal development, the sinus and the sinus related glands became for the first time evident. The ossification of the membranous parts of the lateral nasal wall was initiated. In the next two developing stages the maturation of the cartilage, the increasing ossification in the membrane, and the further differentiation of the glands were the major morphological changes in the formation of the lateral nasal wall.     

Catecholamines in regulation of development of GnRH neurons of rat fetuses

The contents of dopamine, serotonin, and noradrenaline in rat fetuses developing under the conditions of their deficiency induced by administration of alpha-methyl-para-tyrosine to females during 11th to 16th or 20th day of pregnancy and in fetuses, whose mothers were given saline at the same time, were determined using HPLC with subsequent electrochemical detection. Administration of alpha-methyl-para-tyrosine led to decreased levels of dopamine and noradrenaline in the areas of migration of GnRH-neurons in fetuses on days 17 and 21 of prenatal development. The concentration of serotonin remained unchanged, except in the head nasal area in males on day 21. The areas of interaction between the brain catecholaminergic systems and migrating and differentiating GnRH-neurons were determined by double immunohistochemical labeling. Close topographical location of GnRH-immunoreactive neurons and tyrosine hydroxylase-immunoreactive in the area of nucleus accumbens on days 17 and 20, as well as in the median eminence on day 20. The GnRH concentration in the caudal areas of migration of GnRH-neurons under the normal conditions and in the case of catecholamine deficiency was determined using radioimmunoassay. After administration of alpha-methyl-para-tyrosine the GnRH concentration in the anterior hypothalamus decreased in females. The data obtained suggest the involvement of catecholamines in the regulation of development of GnRH-Neurons during prenatal development. In addition, the adequacy and efficiency of the used model of catecholamine deficiency for studying the development of such neurons was confirmed.     

Vibrissal inputs into the motor cortex of adult and developing rats

Field potentials (FP) and responses of single neurones to electrical stimulation of vibrissal pads have been recorded in motor cortex in the albino mature and developing rats. The FPs were characterized by 3-phasic shape and high stability in mature rats. The FPs evoked by contralateral stimuli have a range of onset latency of 4 to 24 ms (peak of distribution 8-11 ms); those to ipsilateral stimuli have a latency of 4 to 23 ms (peak of distribution 12-16 ms). Responses of single neurones were evoked with a latency of 9 to 20 ms. Usually, the FPs were evoked by both contralateral and ipsilateral stimulation, and in some tracks were effective only ipsilateral stimuli in the developing rats beginning from the 11th day of life. The FPs in such animals were less stable and more fatigable. During 2-4 weeks of life, FPs evoked by contralateral stimulation appeared with a latency of 15 to 46 ms; during the same period, a latency of single unit responses ranged between 20 to 33 ms. The FPs to ipsilateral stimuli appeared with a latency of 18 to 47 ms, a latency of single unit responses of 27 to 47 ms. The results indicate functional immaturity of vibrissal system up to the end of the first month of rat life.     

Distribution of histamine in the rat kidney during pregnancy and development

Summary An antiserum against conjugated histamine and two oligonucleotide probes that detect the mRNA encoding L-histidine decarboxylase (HDC) involved in histamine synthesis were used to study the appearance of histamine and its location in the kidneys of fetal, newborn and young postnatal rats and in the kidneys of pregnant rats. On embryonic days 16 and 18 (E16 and E18), some HA-immunoreactive (HA-ir) cells were found within the largest S-shaped bodies. Histamine was found to appear rapidly between the 18th and 20th embryonic days in the convoluted tubules of the kidneys. On postnatal day 0 (P0), the distal convoluted tubules and collecting ducts exhibited bright fluorescence, the intensity of which decreased quickly so that it was faint on day P4 and absent at later stages. In kidneys of pregnant rats HA-ir was found in the epithelium of both the Bowman's capsule, collecting ducts and in a few cells within the tubules. Nonuniform HA-ir was also detected within glomeruli. No evidence for the presence of L-histidine decarboxylase mRNA in kidneys of fetuses or pregnant rats was seen. It is concluded that distinct structures in the developing rat kidney contain histamine during a period around birth from day E20 to day P4. In the pregnant rat, the epithelium that is in direct contact with the urine flow is immunoreactive for histamine from day 16 to 20 of pregnancy. The results suggest that histamine is not synthesized locally in the kidneys but rather originates from other tissues.     

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.     

Profile of acetylcholinesterase in brain areas of male and female rats of adult and old age

Inhibition of acetylcholinesterase (AChE)-metabolizing enzyme of acetylcholine, is presently the most important therapeutic target for development of cognitive enhancers. However, AChE activity in brain has not been properly evaluated on the basis of age and sex. In the present study, AChE activity was investigated in different brain areas in male and female Sprague-Dawley rats of adult (3 months) and old (18-22 months) age. AChE was assayed spectrophotometrically by modified Ellman's method. Specific activity (micromoles/min/mg of protein) of AChE was assayed in salt soluble (SS) and detergent soluble (DS) fractions of various brain areas, which consists of predominantly G1 and G4 molecular isoforms of AChE respectively. The old male rats showed a decrease (40-55%) in AChE activity in frontal cortex, striatum, hypothalamus and pons in DS fraction and there was no change in SS fraction in comparison to adult rats. In the old female rats the activity was decreased (25-40%) in frontal cortex, cerebral cortex, striatum, thalamus, cerebellum and medulla in DS fraction whereas in SS fraction the activity was decreased only in hypothalamus as compared to adult. On comparing with old male rats, old female rats showed increase in AChE activity in cerebral cortex, hippocampus and hypothalamus of DS fraction and decrease in hypothalamus of SS fraction. There was a significant increase in AChE activity in DS fraction of cerebral cortex, hippocampus, hypothalamus, thalamus and cerebellum in female as compared to male adult rats. However, no significant change in AChE activity was found in the SS fraction, except hypothalamus between these groups. Thus it appears that age alters AChE activity in different brain regions predominantly in DS fraction (G4 isoform) that may vary in male and female. These observations have significant relevance to age related cognitive deficits and its pharmacotherapy.     

Long-term changes in the activity of Purkinje cells and efferent cerebellar neurons following bilateral destruction of the inferior olive

The spontaneous discharge frequency of Purkinje cells and neurones of the cerebellar nuclei was evaluated in rats after complete bilateral destruction of their inferior olive with 3-acetylpyridine, performed one day to six months before. The deafferentation from the climbing fibers produced an increased inhibitory action of the Purkinje cells on their target neurones, lasting at least for one week. A relative compensation took place progressively during the first month, but the normal activity of the circuit did not recover even after six months.