Histochemical and chemical studies on pre- and postnatal development of the different systems of “short” and “long” adrenergic neurons in peripheral organs of the rat

Summary The adrenergic innervation in the submaxillary gland, heart, kidney, small intestine, and accessory male genital organs and the development of the adrenal chromaffin cells and the sympathetic ganglia were studied in the rat from 15 days post coitum to 16 days post partum using the fluorescence histochemical method of Falck and Hillarp. The postnatal development of the noradrenaline concentrations in the heart and vas deferens was followed by fluorometric determinations.At about 15 days post coitum, the anlagen of the sympathetic chains were well visible in the form of two dorsal segmented columns of small branching sympathicoblasts exhibiting an intense catecholamine fluorescence. In the midline, ventrally to these two anlagen, another column of sympathicoblasts developed; this seemed to give rise to the prevertebral ganglia and to the short adrenergic neurons supplying the internal genital organs. At the level of the adrenal anlagen, small intensely fluorescent chromaffin cells were collected in two bilateral groups which became enclosed by adreno-cortical cells. This enclosure was, however, not complete even at two weeks post partum.Bundles of growing sympathetic nerves were visible in the periphery of the various organs studied at 19–21 days post coitum. A terminal innervation of the organs suggestive of a functional transmitter mechanism did not start to establish until at or immediately after birth. The final pattern of innervation was usually reached at about one week post partum, and the following development proceeded largely in the form of a quantitative increase in the number of nerves participating in the innervation apparatus. The adult level of noradrenaline in the heart and vas deferens was reached three to five weeks after birth. The small intestine was an exception in that the final pattern of innervation in the wall was attained immediately after birth.There was no overt difference in the rate of development of the terminal sympathetic innervation in organs supplied by short adrenergic neurons (accessory male genital organs) compared to the innervation of the submaxillary gland, heart and kidney, which receive classical long adrenergic neurons.The work was supported by a grant from the Association for the Aid of Crippled Children, New York, and was carried out within a research organization sponsored by the Swedish Medical Research Council (grants No. B71-14X-56-07A and B71-14X-712-06A).     

Dependence of the cytotoxic effect of guanethidine on the degree of sympathetic activity

Young rats aged 15-29 days received a subcutaneous injection of guanethidine sulphate (5 mg/kg body weight) every day. Owing to damage to the postganglionic sympathetic neurones, on about the 60th day of life we observed a significant decrease in the noradrenaline concentration in these animals' hearts compared with the controls. If every guanethidine injection was followed immediately by intensive physical exercise, there was no drop in the heart noradrenaline concentration. Physical exercise of the same intensity performed a few hours before injecting guanethidine did not prevent the drop in the noradrenaline concentration in the heart. The results show that an exercise-induced increase in sympathetic activity, at a time when guanethidine is circulating in the blood and accumulating in the adrenergic neurones, inhibits the cytotoxic effect of guanethidine. Isolated physical exercise performed between the 15th and 29th day of life leads to an increase in the noradrenaline content of the heart of rats aged 60 days.     

Disorder of catecholamine synthesis in early ontogeny and hypophyseal-adrenal cortical function in adult rats

The effects of daily intraperitoneal (150 mg/kg of body weight) injections of alpha-methyl-dl-tyrosine (MT), an inhibitor of tyrosine hydroxylase, on the 2nd to 4th, 5th to 7th or 10th to 12th days of life on the pituitary-adrenal function and brain adrenaline level in 3 to 4-month old rats were studied. MT treatment on the 5th to 7th days resulted in a decrease of noradrenaline content in hypothalamus and midbrain and chronic decrease of basal corticosterone level in blood, its diurnal fluctuations being preserved. MT injections on the 10th to 12th days were accompanied by a decrease of the basal corticosterone level, but the brain noradrenaline level remained unchanged. A study of pituitary-adrenal stress reactivity of adult rats has revealed no specific MT effect. A conclusion was drawn that the MT treatment applied exerted a long-term effect, predominantly, on the regulation of tonic corticosterone secretion.     

Increase of noradrenaline contents in the hypothalamus of neonatal female rats under the effects of 4-hydroxyestradiol-17beta

Catecholamine content was studied in hypothalamus of neonatal Wistar female rats treated with 4-hydroxyestradiol-17 (4-OH-E2) in a dose of 10 mg for 1-5 days of life. 4-OH-E2 induced a reliable increase in hypothalamic noradrenaline level in 24 h after the last injection, but not on the 7th, 10th or 12th postnatal days. There was no change in dopamine level. We have postulated a relationship between the increase in hypothalamic noradrenaline content induced by 4-OH-E2 and defeminization effects of 4-OH-E2 on the developing brain of female rats.     

Post-natal development of brown adipose tissue in the rat bred at 23 degrees C or 28 degrees C.

In view to study the effects of thermal environment on the development and the thermogenic activity of interscapular brown adipose tissue (BAT), young rats born at 23 degrees C or 28 degrees C were sacrificed at 1, 3, 7, 11, 14 or 21 days after birth. The rate of increase in animal weight was quite the same at both temperatures up to the 14th day. The development of BAT and its contents in lipids, in water and in noradrenaline indicate that the energetic activity of the tissue is greatly stimulated in rats kept at 23 degrees C up to the 11th day. It is concluded that in rats bred in the habitual thermal conditions (23 degrees C), the occurrence of non shivering thermogenesis (NST) is important during the period of ten days after birth; in the following period NST could be progressively replaced by other thermoregulatory processes.     

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.     

Clonogenic hemopoietic cells (CFU-S) in mouse pre- and postnatal ontogeny

Content of three classes of clonogenic haemopoietic cells (CFU-S-7, CFU-S-11 and CFU-S-ep) was determined in haemopoietic organs of mouse during embryogenesis (10, 14 and 18 day) and postnatal ontogenesis (2, 3 and 7 day, 1, 2, 3 and 18 month). CFU-S-7 and CFU-S-11 that from big splenic colonies on 7th and 11th days of transplantation are present in liver, spleen and bone marrow at all developmental stages. However their concentration and CFU-S-7 CFU-S-11 ratio change in haemopoietic organs. CFU-S-ep that form small colonies on 11th day are observed before birth in liver and spleen and 1 week after birth there and also in bone marrow but are practically absent from haemopoietic organs of older animals. Thus, CFU-S compartment structure is characterized by definite ratio of its subpopulations. It seems to reflect functional state of haemopoietic system during development.     

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.     

Development of prejunctional alpha 2 adrenergic receptor mediated feedback control of the pressor response to sympathetic nerve stimulation in hypertensive and normotensive rats

Development of prejunctional alpha 2 adrenergic receptor inhibition of pressor responses to sympathetic nerve stimulation in the spontaneously hypertensive Wistar-Kyoto rat was compared with genetically similar (Wistar-Kyoto) and different (Sprague-Dawley) normotensive rats. The sympathetic outflow was stimulated at frequencies of 1 to 20 Hz in pithed rats at 10,14,20 and 60 days of age. The antagonist, rauwolscine was given to block alpha 2 mediated feedback inhibition of noradrenaline release and the incidence of enhanced pressor responses determined. In Sprague-Dawley but not in spontaneously hypertensive or Wistar-Kyoto rats the changes in the incidence of enhanced responses parallel development of indices of sympathetic activity in other studies of the rat. Thus at 10 days of age (when activity is low), the incidence of rauwolscine-enhanced responses was 45%; at 14 days, (coinciding with onset of baroreflex control), incidence fell to 14%; in the 3rd postnatal week (when there is sympathetic hyperactivity), incidence increased to greater than 90%; finally, incidence, like activity declined in adults. In Wistar-Kyoto rats, the incidence of enhanced responses was like the other strains at 10 days but then decreased during development. In spontaneously hypertensive rats, enhanced responses were also less evident during week 3 and greatly diminished in adults. We conclude that in the spontaneously hypertensive and normotensive variants of Wistar-Kyoto strain rats the limit of alpha 2 adrenergic receptor feedback control of noradrenaline release is reached prematurely and is attenuated relative to the level of neuronal activity. In keeping with this hypothesis, the basal rate of noradrenaline utilization (measured in kidney) was higher at 20 days in Wistar-Kyoto than in Sprague-Dawley, but Sprague-Dawley showed greater enhancement of noradrenaline level and utilization after rauwolscine. Thus, the limitation to feedback control may be in development of prejunctional alpha 2 adrenergic receptors and/or their coupling to transmitter synthesis and release. Attenuated prejunctional alpha 2 adrenergic receptor inhibition is not linked obligatorily to development of hypertension in the spontaneously hypertensive Wistar-Kyoto rat.     

Catecholamines and DOPA in the amniotic fluid and amnion of the chick embryo

Identification and quantitative fluorimetric assay have been made on the content of DOPA, dopamine, noradrenaline and adrenaline in the amniotic fluid and amnion of the developing chick embryos. Significant increase in the content of DOPA, noradrenaline and adrenaline in the amniotic fluid was observed between the 6th and the 13th days of incubation; dopamine content sharply decreases at the 13th day. The content on amines in the amnion tissue remained essentially constant throughout the investigated period. The role of catecholamine in amniotic fluid in regulation of contractile activity of amniotic membrane in the developing chick embryo is discussed.     

Postnatal development of chronotropic responses to chemical and electrical stimulation of adrenergic nerve endings in the sinoatrial node of the heart of the albino rat

The positive chronotropic response to stimulation of adrenergic nerve endings in the sinoatrial node was studied in isolated atria from the hearts of rats of different ages. Dimethylphenylpiperazinium (DMPP) was used for chemical stimulation and transmural stimulation of the sinoatrial node region as electrical stimulation; in both cases noradrenaline is released from the nerve endings. With both stimulation methods, postnatal development was recorded in two phases. In the first phase, positive chronotropic responses are markedly increased and attained the maximum at the age of 14 days on using DMPP and of 24 days on using electrical stimulation. In the second phase, positive chronotropic responses diminish and at the age of about 45 days, with both stimulation methods, they become reduced to adult level. The first developmental phase can be attributed to an increase in the noradrenaline content of the nerve endings and the release of a larger amount of the transmitter during stimulation, together with an increase in the noradrenaline sensitivity of the cells of the sinoatrial node. It is not clear why positive chronotropic responses decrease in the second phase, when the noradrenaline content of the myocardial tissue continues to rise and pacemaker sensitivity to noradrenaline is not reduced.     

Portal and arterial free and conjugated noradrenaline in two models of portal hypertension in rats

Free and conjugated noradrenaline concentrations were measured in portal-venous and arterial plasma from sham-operated rats or rats with portal hypertension. Two types of portal hypertension in rats were evaluated: in portal vein stenosed rats, the liver was normal, whereas cirrhosis developed in bile duct ligated rats. In cirrhotic rats, arterial free noradrenaline level was higher than in both sham-operated and portal-stenosed rats, this indicating that enhanced sympathetic nervous activity depends on the development of cirrhosis. In all groups of rats, portal venous plasma free noradrenaline was higher than arterial plasma level, indicating a production of noradrenaline by splanchnic organs. Arterial noradrenaline level may be mainly dependent on this splanchnic production in case of portal hypertension. Sulfoconjugated and glucuronoconjugated noradrenaline plasma levels were similar in the three groups of rats. This shows that alteration in conjugation is not likely to be a major factor in the abnormal circulating levels of free noradrenaline observed in cirrhotic rats.     

Effect of thyroidectomy on cardiovascular responses to hypoxia and tyramine infusion in fetal sheep

Fetal sheep were thyroidectomized at 80 days' gestation and reoperated at 118-122 days for insertion of vascular catheters. The effects of hypoxaemia and intravenous tyramine infusion on plasma catecholamine concentrations, blood pressure and heart rate were then determined in experiments at 125-135 days' gestation. Age matched intact fetuses were also studied. Thyroidectomy was associated with increased concentrations of noradrenaline, adrenaline and dopamine in some thoracic and abdominal organs, increased noradrenaline concentrations in the cerebellum, and decreased adrenaline concentrations in the hypothalamus, cervical spinal cord, and superior cervical and inferior mesenteric ganglia. Arterial pressure was significantly lower in the thyroidectomized fetuses (34.0 +/- 0.15 mmHg) than in intact fetuses (44.7 +/- 0.2 mmHg; p less than 0.001). In contrast, plasma noradrenaline concentrations were significantly higher in the thyroidectomized fetuses (2.04 +/- 0.25 ng/ml) compared to the intact fetuses (0.99 +/- 0.08 ng/ml; P less than 0.001). In the intact fetuses there was a significant increase in plasma noradrenaline concentration and blood pressure during hypoxaemia, and bradycardia at the onset of hypoxaemia. In contrast, in the thyroidectomized fetuses hypoxaemia did not cause significant change in plasma catecholamine concentrations, blood pressure or heart rate. Infusion of tyramine produced a 1.9-fold increase of plasma noradrenaline in thyroidectomized fetuses compared to a 9.2-fold increase in the intact fetuses (P less than 0.05). Tyramine infusion caused a similar proportional increase of blood pressure in both thyroidectomized and intact fetuses. Heart rate decreased during the tyramine-induced hypertension in the intact fetus, but increased in the thyroidectomized fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphologic and hormonal correlates of true hermaphroditism in the rabbit

A macro-microscopical and histological investigation of the urogenital system organs of a rabbit-hermophrodite has been performed with an aim to compare the content of sex steroids in the peripheral and in the gonadal blood estimated by means of radioimmunological methods. Certain anomalies have been found out in the development of external and internal sex organs, kidneys and urinary bladder. Testosterone and estradiol concentration in blood flowing from the ovotestis is decreased, as it is in the periphral blood. The rabbit urogenital system organs have been studied from the 12th day of the intrauterine development up to the 10th day of the postnatal life. The results of the investigation performed make it possible to consider that the developmental anomalies in the rabbit-hermophrodite have, evidently, appeared between the 20th--25th days of its intrauterine development as a result of certain disbalance of sex steroids.     

Effect of repeated exercise on postnatal development of the noradrenaline content of the albino rat heart, spleen and skeletal muscle

The authors studied the effect of repeated elevation of sympathetic activity on the postnatal development of the noradrenaline content of tissues of the albino rat. Between the ages of 15 and 29 days, young rats were forced to swim in water heated to 25 degrees C, 3 X 30 min on weekdays and 1 X 30 min on Saturdays and Sundays. At 30, 45 and 65 days, the noradrenaline content of the tissues was determined spectrofluorometricaLly by the trihydroxyindole method. The noradrenaline content of the heart of trained rats was higher than in the controls in all the given age groups and the size of the absolute difference rose with advancing age. The noradrenaline content of the spleen developed similarly. Repeated exercise did not lead to an increase in the noradrenaline content of skeletal muscle. The results show that the repeated elevation of the activity of sympathetic adrenergic neurones which occurs in young rats during exercise is a long-term factor stimulating the development of sympathetic innervation of the heart and spleen. The development of the neurones innervating skeletal muscle was not stimulated, probably because the activity of these neurones is not increased by stress.     

The noradrenaline content and innervation of brown adipose tissue in the young rabbit

This work examined the noradrenaline content of brown adipose tissue, the metabolic response to endogenous noradrenaline released during tyramine infusion, and the innervation of brown fat at the electron microscopic level in the young rabbit. The noradrenaline content (ng/g) of the interscapular and cervical fat deposits ranged from 256 +/- 51 to 343 +/- 59 and 399 +/- 18 to 694 +/- 92, respectively, in four groups of rabbits (1-2, 7-8, 12-13, and 25-27 days of age). There was considerable variation amongst animals in each age group, but no evidence of a major increase or decrease in noradrenaline content during the first 4 weeks of life. Intravenous infusion of tyramine (100 micrograms X kg-1 X min-1) increased plasma noradrenaline concentration, oxygen consumption, and blood flow to brown fat. Thus noradrenaline released from endogenous sites, as well as injected noradrenaline, will initiate the thermogenic response of brown fat. Ultrastructurally, unmyelinated axons that were not organized in a fascicle were observed adjacent to the adipocytes in the late gestation fetus. By 1 week of age of axons were surrounded by Schwann cell cytoplasm which formed a fascicle. However, no evidence of myelination was found up to 21 days of age. Collectively, the data indicate that the brown adipocyte is fully responsive at 1-2 days of age even though myelination of the nerves is incomplete, and that the incomplete development of the sympathetic nerves at birth is not a factor in the synthesis of noradrenaline in the very young rabbit. In addition, brown fat of the newborn rabbit is not as thermogenically active as the brown fat of the cold-acclimated rat.     

Distribution of radioactivity in subcellular fractions of genital tract tissue and skeletal muscles of guinea pig embryos after administration of 1 alpha,2 alpha-3H(n) testosterone in vitro

The dynamics of radioactivity in subcellular fractions of the reproductive system and muscle has been studied in the guinea pig embryos from the 21st day of development till birth after the preincubation in a medium containing 1 alpha,2 alpha-3H(n) testosterone (T-3H). The total uptake of T-3H per mg tissue is higher in the reproductive system than in the muscle and attains the highest values during the period of differentiation of target organs by the male type (30-33 days). The nucleocytoplasmic ratio of radioactivity is shifted towards its increase in the nucleus with maximum on the 41st-45th days and by the moment of birth. Radioactivity in the microsomal fraction is higher than in the cytosol (soluble proteins). From the 27th day of development on, when the hormone uptake becomes selective, the radioactivity in the cytosol from the reproductive organs is higher than in the muscle tissue. The data obtained suggest the importance of processes in microsomes for differentiation and organogenesis of reproductive system.     

Brain monoamines and the hypophyseo-gonadal system of adult rats after the androgenization of their mothers during pregnancy

The brain noradrenaline system and its involvement in the control of the hypothalamic-hypophysial-gonadal complex were studied on 3 month old Wistar rats, which descended from the mothers treated with testosterone propionate on the 16th and 18th day of pregnancy. A season-independent weakening of the activating effect of noradrenaline, introduced into the brain lateral ventricle on the estradiol and testosterone content of the peripheral blood has been shown, compared with the descendants of the mothers treated with oil at the same times of pregnancy. Unlike noradrenaline, the intraventricular introduction of serotonin or dopamine was accompanied by a similar activation of the hypophysial-gonadal system in the descendants from both the androgenized and control mothers. In the descendants from the former, the thermal reaction to the intraventricular introduction of noradrenaline underwent changes as well. A conclusion is reached that testosterone propionate, introduced during the prenatal period, exerts a long-term and to a certain extent, selective and generalized effect on the developing brain noradrenaline system. In the rats, the critical period for the disturbance in androgen balance influence on the noradrenaline mechanisms of brain is timed to no later than the 18th day of prenatal development.     

The effect of ethanol exposure in pregnancy on maturation of monoaminergic systems in the developing rat bran

Simultaneous study of the main neurotransmitter of monoaminergic system of the brain, its metabolites, activity of catechol-O-methyltransferase (COMT) and the state of different subtypes of dopamine (DA) receptors in the developing brain of offspring from mothers alcoholized in gestation and feeding periods revealed a decrease in activity of all monoaminergic systems studied with reduction of noradrenaline and DA level in alcoholized fetus as well as of mPNA of COMT, an enzyme of catecholamine metabolism, in the structures of the forebrain on the 17th day but not on 13th day of prenatal development. In parallel experiments, an increase of the contents of both long and short splice variants of D2 DA receptor was registered. In postnatal period (days 4, 10, 17), further decrease of the DA system activity was observed, particularly a reduction of DOPAC level and DOPAC/DA ratio in rat litter, mothers of whom took alcohol in the gestation period with withdrawal it after birth of offspring. The serotonin system activity was also reduced in alcoholized litter in the postnatal period and was registered in the early stages (on the 4th day of life). Therefore, the serotonin system activity is changing at early stages of development (the 4th day), whereas inhibition of the DA system activity is registered at later stages (the 10th day of life).