Effect of catecholamines on migration and differentiation of gonadotropin releasing hormone-neurons in rats

The GnRH producing neurons are the key link of neuroendocrine regulation of the adult reproductive system. Synthesis and secretion of GnRH are, in turn, under the afferent catecholaminergic control. Taking into account that catecholamines exert morphogenetic effects on target cells during ontogenesis, this study was aimed at investigation of the effects of catecholamines on development of GnRH neurons in rats during ontogenesis. We carried out comparative quantitative and semiquantitative analyses of differentiation and migration of GnRH neurons in fetuses of both sexes under the conditions of normal metabolism of catecholamines (administration of saline) or their pharmacologically induced deficiency (administration of alpha-methylparatyrosine). The inhibition of catecholamine synthesis from day 11 of embryogenesis led to an increasing number of GnRH neurons in rostral regions of the trajectory of their migration over the brain: in the area of olfactory tubercles on day 17 and in the area of olfactory bulb on days 18 and 21. In addition, the optical density of GnRH neurons located in the rostral regions of migration was higher in the fetuses after administration of alpha-methylparatyrosine during embryogenesis, as compared to the control. It has been concluded that catecholamines stimulate the migration of GnRH neurons and affect their differentiation.     

Influence of Catecholamines on Migration and Differentiation of GnRH Neurons in Rats

The GnRH producing neurons are the key link of neuroendocrine regulation of the adult reproductive system. Synthesis and secretion of GnRH are, in turn, under the afferent catecholaminergic control. Taking into account that catecholamines exert morphogenetic effects on target cells during ontogenesis, this study was aimed at investigation of the effects of catecholamines on development of GnRH neurons in rats during ontogenesis. We carried out comparative quantitative and semiquantitative analyses of differentiation and migration of GnRH neurons in fetuses of both sexes under the conditions of normal metabolism of catecholamines (administration of saline) or their pharmacologically induced deficiency (administration of -methyl-para-tyrosine). The inhibition of catecholamine synthesis from day 11 of embryogenesis led to an increasing number of GnRH neurons in rostral regions of the trajectory of their migration over the brain: in the area of olfactory tubercles on day 17 and in the area of olfactory bulb on days 18 and 21. In addition, the optical density of GnRH neurons located in the rostral regions of migration was higher in the fetuses after administration of -methyl-para-tyrosine during embryogenesis, as compared to the control. It has been concluded that catecholamines stimulate the migration of GnRH neurons and affect their differentiation.     

Effect of serotonin on differentiation of neurons producing vasoactive intestinal polypeptide in the rat suprachiasmatic nucleus

This study was aimed to evaluate the morphogenetic influence of serotonin on the differentiating vasoactive intestinal polypeptide (VIP) neurons of the suprachiasmatic nucleus (SCN) in rats. The comparative morpho-functional analysis of VIP neurons was made in adult rats which developed under normal metabolism of serotonin or in its deficiency. The serotonin deficiency was induced in foetuses by injections of p-chlorophenilalanine to pregnant mothers. Control rats received the saline. According to our data, there was no difference in the VIP mRNA concentration and most probably in VIP synthesis in the SCN in adult rats following prenatal serotonin depletion compared to the control. However, the serotonin deficiency resulted in increase of the VIP concentration in cell bodies and of the VIP neurones number. The size of the VIP-neurones did not change in pharmacologically treated rats compared to the controls showing no functional hypertrophy of the neurones as a result of the activation of specific syntheses. From the above data, it follows that serotonin provides an imprinting influence differentiating the VIP neurones, contributing most probably to development of the VIP release mechanism.     

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.     

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 α-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 α-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 α-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.     

Maternal dexamethasone exposure during pregnancy in rats disrupts gonadotropin-releasing hormone neuronal development in the offspring

The migration of gonadotropin-releasing hormone (GnRH) neurons from the olfactory placode to the preoptic area (POA) from embryonic day 13 is important for successful reproduction during adulthood. Whether maternal glucocorticoid exposure alters GnRH neuronal morphology and number in the offspring is unknown. This study determines the effect of maternal dexamethasone (DEX) exposure on enhanced green fluorescent protein (EGFP) driven by GnRH promoter neurons (TG-GnRH) in transgenic rats dual-labelled with GnRH immunofluorescence (IF-GnRH). The TG-GnRH neurons were examined in intact male and female rats at different postnatal ages, as a marker for GnRH promoter activity. Pregnant females were subcutaneously injected with DEX (0.1 mg/kg) or vehicle daily during gestation days 13–20 to examine the number of GnRH neurons in P0 male offspring. The total number of TG-GnRH neurons and TG-GnRH/IF-GnRH neuronal ratio increased from P0 and P5 stages to P47–52 stages, suggesting temporal regulation of GnRH promoter activity during postnatal development in intact rats. In DEX-treated P0 males, the number of IF-GnRH neurons decreased within the medial septum, organum vasculosom of the lamina terminalis (OVLT) and anterior hypothalamus. The percentage of TG-GnRH neurons with branched dendritic structures decreased in the OVLT of DEX-P0 males. These results suggest that maternal DEX exposure affects the number and dendritic development of early postnatal GnRH neurons in the OVLT/POA, which may lead to altered reproductive functions in adults.     

Migration and differentiation of gonadotropin-releasing hormone-producing neurons in the brain of mouse fetus exposed to excess of serotonin

The work has been carried out on mice of the Tg8 line with knockout of gene of monoamineoxidase A with an increase of serotonin and noradrenaline content in the brain, and on mice of the C3H line with unchanged genome and normal concentration of monoamines. An immunocytochemical study has been performed of development of neurons producing gonadotropin-releasing hormone (GnRH) under conditions of excess of serotonin and noradrenaline in the mice in embryogenesis. The GnRH-neurons were revealed at the 18th day of embryonic development in telencephalon along trajectory of their migration from olfactory bulbs to the retrochiasmatic area. In telencephalon of mouse embryos of the Tg8 line, a redistribution of the GnRH-neurons along their migration trajectory was observed as compared with embryos of the C3H line mice. The percent of the GnRH-neurons in the Tg8 mouse embryos in caudal parts of the migration trajectory was lower than in rostral parts, the opposite distribution of the neurons being observed in the C3H line mouse embryos; at the excess of serotonin and noradrenaline in the Tg8 line mouse embryos, the total amount of GnRH-neurons in the brain was lower than in the C3H mice. In males of the Tg8 line mice under conditions of excess of serotonin and noradrenaline the optical density of neurons, which correlated with the GnRH concentration in the cell, was higher than in control mice. Thus, in the Tg8 mice under conditions of the serotonin and noradrenaline excess, migration of the GnRH-neurons to their final anlage in hypothalamus is accelerated as well as the total number of the GnRH-neurons decreases, which indicates a decrease of proliferation of cells-precursors and the earlier differentiation of neurons.     

Migration and differentiation of neurons producing gonadotropin-releasing hormone under conditions of serotonin excess in the brain of mouse embryos

The work has been carried out on mice of the Tg8 line with knockout of gene of monoamineoxidase A with an increase of serotonin and noradrenaline content in the brain, and on mice of the C3H line with unchanged genome and normal concentration of monoamines. An immunocytochemical study has been performed of development of neurons producing gonadotropin-releasing hormone (GnRH) under conditions of excess of serotonin and noradrenaline in the mice in embryogenesis. The GnRH-neurons were revealed at the 18th day of embryonic development in telencephalon along trajectory of their migration from olfactory bulbs to the retrochiasmatic area. In telencephalon of mouse embryos of the Tg8 line, a redistribution of the GnRH-neurons along their migration trajectory was observed as compared with embryos of the C3H line mice. The percent of the GnRH-neurons in the Tg8 mouse embryos in caudal parts of the migration trajectory was lower than in rostral parts, the opposite distribution of the neurons being observed in the C3H line mouse embryos; at the excess of serotonin and noradrenaline in the Tg8 line mouse embryos, the total amount of GnRH-neurons in the brain was lower than in the C3H mice. In males of the Tg8 line mice under conditions of excess of serotonin and noradrenaline the optical density of neurons, which correlated with the GnRH concentration in the cell, was higher than in control mice. Thus, in the Tg8 mice under conditions of the serotonin and noradrenaline excess, migration of the GnRH-neurons to their final anlage in hypothalamus is accelerated as well as the total number of the GnRH-neurons decreases, which indicates a decrease of proliferation of cells-precursors and the earlier differentiation of neurons.     

Perinatal development of mammillotegmental connections in rats

Development of direct axonal connections of the hypothalamic mammillary bodies with ventral and dorsal tegmental nuclei of Gudden was studied on fixed rat brains from day 14 of embryonic development until day 10 of postnatal development using the method of diffusion of the lipophilic fluorescent carbocyanine tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate along the neuronal membranes. The tracer was inserted into the mammillary bodies or into the tegmentum and, after incubation in a fixative, fluorescent nerve cells and nerve fibers were visualized in the brain tissue. The mammillotegmental tract was found to start developing earlier than other projection systems of the mammillary bodies. On days 14–15 of embryonic development, it was visualized as a bundle of axons running from the mammillary bodies caudally to the midbrain. A group of neurons in the midbrain tegmentum and their axons going to the mammillary bodies via the mammillary peduncle were first visualized on day 19 of embryonic development. The mammillotegmental tract and mammillary peduncle developed progressively from the moment of birth. Ventral and dorsal tegmental nuclei were formed in the midbrain by day 10 of the postnatal development. Thus, the formation of reciprocal connections of the mammillary bodies with midbrain tegmental nuclei was first described during perinatal development in rats.     

Perinatal development of mammillotegmental connections in rats

Development of direct axonal connections of the hypothalamic mammillary bodies with ventral and dorsal tegmental nuclei of Gudden was studied on fixed rat brains from day 14 of embryonic development until day 10 of postnatal development using the method of diffusion of the lipophilic fluorescent carbocyanine tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate along the neuronal membranes. The tracer was inserted into the mammillary bodies or into the tegmentum and after incubation in a fixative fluorescent nerve cells and nerve fibers were visualized in the brain tissue. The mammillotegmental tract was found to start developing earlier than other conducting systems of the mammillary bodies. On days 14-15 of embryonic development, it was visualized as a bundle of axons running from the mammillary bodies caudally to the midbrain. A group of neurons in the midbrain tegmentum and their axons going to the mammillary bodies via the mammillary peduncle were first visualized on day 19 of embryonic development. The mammillotegmental tract and mammillary peduncle developed progressively from the moment of birth. Ventral and dorsal tegmental nuclei were formed in the midbrain by day 10 of the postnatal development. Thus, the formation of reciprocal connections of the mammillary bodies with midbrain tegmental nuclei was first described during perinatal development in rats.     

Increased collagen metabolism in granulomata induced in rats deficient in endogenous prostaglandin precursors

Collagen metabolism was measured (in terms of various hydroxyproline (HP), DNA and protein ratios) in granulomata obtained after s.c. implantation of carrageenan-impregnated and untreated polyether sponges into normal and essential fatty acid deficient (EFAD) rats for 8 and 15 days. Collagen synthesis (HP/protein) in day 8 and 15 untreated granulomata was the same for both normal and EFAD rats, though collagen breakdown (total HP) appeared to be greater in EFAD granulomata on day 15. With carrageenan-impregnated sponges, collagen synthesis in EFAD granulomata was much greater than in normal granulomata on both day 8 and day 15. Ratios of protein and/or HP to DNA (probably indicative of cellular infiltration) were increased in EFAD rats with both sponge types, though this increase was less pronounced with carrageenan-impregnated sponges. It is suggested that endogenous prostaglandin (PG) production (marledly reduced during EFA deficiency) may exert a negative feedback effect on collagen metabolism during proliferative inflammation.     

Increased collagen metabolism in granulomata induced in rats deficient in endogenous prostaglandin precursors

Collagen metabolism was measured (in terms of various hydroxyproline (HP), DNA and protein ratios) in granulomata obtained after s.c. implantation of carrageenan-impregnated and untreated polyether sponges into normal and essential fatty acid deficient (EFAD) rats for 8 and 15 days. Collagen synthesis (HP/protein) in day 8 and 15 untreated granulomata was the same for both normal and EFAD rats, though collagen breakdown (total HP) appeared to be greater in EFAD granulomata on day 15. With carrageenan-impregnated sponges, collagen synthesis in EFAD granulomata was much greater than in normal granulomata on both day 8 and day 15. Ratios of protein and/or HP to DNA (probably indicative of cellular infiltration) were increased in EFAD rats with both sponge types, though this increase was less pronounced with carrageenan-impregnated sponges. Is is suggested that endogenous prostaglandin (PG) production (markedly reduced during EFA deficiency) may exert a negative feedback effect on collagen metabolism during proliferative inflammation.     

Study of serotonin effects on histogenesis of embryonic rat neocortex on a model of ectopic neurotransplantation

A comparative study of dynamics of development of ectopic grafts of the embryonic (E14) neocortical anlagen obtained from intact rats, rats injected with serotonin inhibitor para-chlorophe-nylalanine (PCPA) on the 11th day of pregnancy as well as the same anlagen incubated before grafting in the serotonin-containing medium. The aim of the study was to reveal influence of serotonin on division and differentiation of the embryonic neocortical cells. Investigation of degeneration, mitotic activity and differentiation (using immunohistochemical revealing of the nuclear protein of nervous cells (NeuN)) of the grafted cells demonstrated contribution of serotonin to survival and differentiation of neuroepithelial cells of the grafts and also to regulation of their proliferation. Serotonin is supposed to have a stimulatory effect on the rate of cellular cycle of the grafted cells, thereby accelerating differentiation of neurons.     

NADPH-diaphorase-positive nerve cells in heterotopic spinal cord transplants

The method of ectopic transplantation of embryonic CNS rudiments makes it possible to study the mechanisms underlying adaptation of the transplanted embryonic rudiments. The production of nitric oxide by cells is considered as one of such mechanisms. NADPH-diaphorase is an index of the presence of nitric oxide synthase in cells. It was shown that the nerve cells of rat embryonic spinal cord transplants preserved their capacity to express NADPH-diaphorase after transplantation in the sciatic nerve of an adult animal for six months. The dynamics of NADPH-diaphorase-positive neurons of rat embryonic spinal cord developing after transplantation and in situ were studied. In spinal cord neck region, small bipolar NADPH-diaphorase-positive neurons were visualized on day 17 of prenatal development. After transplantation of the embryonic (day 15) spinal cord in the nerve, NADPH-diaphorase-positive neurons were formed later than in situ: within seven days. The results of histochemical studies carried out within six months after the operation suggest a protective role of NADPH-diaphorase in the neurons of allotransplants developing under the conditions of altered microenvironment and insufficient innervation and also suggest that nitric oxide can cause the death of neurons in long surviving transplants.     

Deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and early postnatal lethality

The serotonin (5-HT) system densely innervates many brain areas and is important for proper brain development. To specifically ablate the 5-HT system we generated mutant mice carrying a floxed Munc18-1 gene and Cre recombinase driven by the 5-HT-specific serotonin reuptake transporter (SERT) promoter. The majority of mutant mice died within a few days after birth. Immunohistochemical analysis of brains of these mice showed that initially 5-HT neurons are formed and the cortex is innervated with 5-HT projections. From embryonic day 16 onwards, however, 5-HT neurons started to degenerate and at postnatal day 2 hardly any 5-HT projections were present in the cortex. The 5-HT system of mice heterozygous for the floxed Munc18-1 allele was indistinguishable from control mice. These data show that deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and suggests that the 5-HT system is important for postnatal survival.     

NADPH-Diaphorase-Positive Nerve Cells in Heterotopic Spinal Cord Transplants

The method of ectopic transplantation of embryonic CNS rudiments makes it possible to study the mechanisms underlying adaptation of the transplanted embryonic rudiments. The production of nitric oxide by cells is considered as one of such mechanisms. NADPH-diaphorase is an index of the presence of nitric oxide synthase in cells. It was shown that the nerve cells of rat embryonic spinal cord transplants preserved their capacity to express NADPH-diaphorase after transplantation in the sciatic nerve of an adult animal for six months. The dynamics of NADPH-diaphorase-positive neurons of rat embryonic spinal cord developing after transplantation and in situwere studied. In spinal cord neck region, small bipolar NADPH-diaphorase-positive neurons were visualized on day 17 of prenatal development. After transplantation of the embryonic (day 15) spinal cord in the nerve, NADPH-diaphorase-positive neurons were formed later than in situ: within seven days. The results of histochemical studies carried out within six months after the operation suggest a protective role of NADPH-diaphorase in the neurons of allotransplants developing under the conditions of altered microenvironment and insufficient innervation and also suggest that nitric oxide can cause the death of neurons in long surviving transplants.     

Effect of hypothyroidism on 5-HT1A-, 5-HT2A-receptors and serotonin transporter in the rat brain

Effects of thyroid hormone deficiency on 5-HT1A receptors, 5-HT2A receptors and serotonin transporter in the brain were studied in thyroidectomised Wistar rats receiving an iodine-free diet and receiving 15 micrograms/kg of thyroxine for 21 days. Binding of 3H-8-OH-DPAT to 5-HT1A receptors and 3H-cytalopram to serotonin transporter were unchanged in hypothyroid rats as compared to the control. 3H-ketanserin binding to 5-HT2A receptors was significantly decreased in the frontal cortex in hypothyroid rats. The cortical 3H-ketanserin binding in thyroidectomised rats was normalised after thyroxine replacement. The data suggest that the decrease in the cortical 5-HT2A receptors is the main consequence of impairing effect of hypothyroidism on serotonin neurotransmission.     

Differentiated properties of identified serotonin neurons in dissociated cultures of embryonic rat brain stem

Serotonin neurons in 14-d embryonic rat brain stem were identified by peroxidase-antiperoxidase immunocytochemistry with an affinity-purified antiserotonin antibody. Brain-stem tissue was dissected from 14- or 15- d embryonic rats, dissociated and grown in cell culture for up to 5 wk, and serotonin neurons were identified by immunocytochemistry. Within 24 h of plating, serotonin immunoreactivity was present in 3.3% of neurons. Immunoreactivity in neuronal cell bodies decreased with time, whereas staining of processes increased. The number of serotonin- immunoreactive neurons remained constant at 3-5% over the first 14 d in culture. From 14 to 28 d, the total number of neurons decreased with little change in the number of serotonin neurons, such that, by day 28 in culture, up to 36% of surviving neurons exhibited serotonin immunoreactivity. Similar percentages of cultured brain stem neurons accumulating 3H-serotonin were identified by autoradiography. Uptake was abolished by the serotonin-uptake inhibitor, clomipramine, but was unaffected by excess norepinephrine, or by the norepinephrine-uptake inhibitor, maprotiline. Synthesis of 3H-serotonin was detected after incubation of cultures with 3H-tryptophan, and newly synthesized serotonin was released by potassium depolarization in a calcium- dependent manner. More than 95% of serotonin neurons were destroyed after incubation of cultures with 5,6-dihydroxytryptamine. Brain-stem cultures contained virtually no neurons with the ability to accumulate 3H-norepinephrine or 3H-dopamine. Approximately 40% of brain-stem neurons were labeled with gamma-aminobutyric acid (3H-GABA). However, there was almost no overlap in the surface area of neurons accumulating 3H-serotonin or 3H-GABA.     

Loss of luteinizing hormone surges induced by chronic estradiol is associated with decreased activation of gonadotropin-releasing hormone neurons

Chronic exposure of young ovariectomized rats to elevated circulating estradiol causes loss of steroid-induced LH surges. Such LH surges are associated with cFos-induced activation of GnRH neurons; therefore, we hypothesized that chronic estradiol treatment abolishes LH surges by decreasing activation of GnRH neurons. Regularly cycling rats were ovariectomized and immediately received an estradiol implant or remained untreated. Three days or 2 or 4 wk later, the estradiol-treated rats received vehicle or progesterone at 1200 h, and 7 hourly blood samples were collected for RIA of LH. Thereafter, all rats were perfused, and the brains were examined for immunocytochemical localization of cFos and GnRH. The GnRH neurons from untreated ovariectomized rats rarely expressed cFos. As reported, LH surges induced by 3 days of estradiol treatment were associated with a 30% increase in cFos-containing GnRH neurons, and progesterone enhanced both the amplitude of LH surges and the proportion of cFos-immunopositive GnRH neurons. As hypothesized, the abolition of LH surges caused by 2 or more weeks of estradiol was paralleled by a reduction in the percentage of cFos-containing GnRH neurons, and this effect was delayed by progesterone. These results suggest that chronic estradiol abolishes steroid-induced LH surges in part by inactivating GnRH neurons.