[The development of neuroendocrine regulation in ontogeny]. / Stanovlenie neiroéndokrinnoi reguliatsii v ontogeneze.

Published and author's data on hypothalamic control over the most important endocrine functions during mammalian ontogenesis were reviewed. The data presented indicates that adenohypophyseotropic protein neurohormones are synthesized and accumulated in hypothalamus long before birth. At the end of the prenatal period most of hypothalamic neurohormones reached the adenohypophysis and were involved in regulation of the tropic hormones secretion. Exceptionally, regulative properties of thyroliberin and somatostatin are only manifested in immature-born animals (rats) in early postnatal period.

On the distribution and morpho-functional characteristics of 5-HT-immunoreactive cells in the hypothalamus of fetuses and neonatal rats.

This study attempted to visualize serotonin (5-HT)-immunoreactive (IR) neurons in the hypothalamus of intact fetuses (E18) and neonatal rats (P9) as well as after their pretreatment with some drugs interfering with the 5-HT metabolism and uptake in the serotoninergic neurons (L-tryptophan, pargyline, 5-hydroxytryptophan, fluoxetine). The 5-HT-IR cells were not observed in the hypothalamus of normal, untreated fetuses and neonatal rats. However, two large accumulations of 5-HT-IR neurons appeared in the anterolateral hypothalamus and in the dorsomedial nucleus after the subsequent injections of the monoamine oxidase inhibitor, pargyline, and the amino acid precursor of the 5-HT synthesis, L-tryptophan. A significantly less intensive reaction was observed after injections either of the second precursor of the 5-HT synthesis, 5-hydroxytryptophan instead of L-tryptophan, or pargyline only. Immunostaining, provoked by the pargyline and L-tryptophan pretreatment, was completely blocked by the injection of the specific 5-HT uptake inhibitor, fluoxetine. It means that the 5-HT immunostaining of the hypothalamic neurons may be accounted for by their capacity to take up specifically 5-HT from the environment rather than by its intraneuronal synthesis from L-tryptophan. Nevertheless, the 5-HT synthesis from 5-hydroxytryptophan in these cells cannot be excluded. The uptake of extracellular 5-HT into catecholaminergic neurons can be excluded as nomifensine, the specific inhibitor of the uptake to these neurons, did not modify the immunostaining.

Ontogenesis of tyrosine hydroxylase-immunopositive structures in the rat hypothalamus. An atlas of neuronal cell bodies.

The development of the catecholaminergic system in the hypothalamus and in the septal region was studied in rats from the 12th fetal day until the 9th postnatal day. Catecholaminergic structures were visualized with pre-embedding immunocytochemistry using antiserum to tyrosine hydroxylase. An intensification of diaminobenzidine product with silver and gold was additionally applied to make the immunocytochemical technique more sensitive. In this paper only the data on the appearance and distribution of the tyrosine hydroxylase-immunopositive neurons (cell bodies) are presented, whereas the catecholaminergic innervation of the hypothalamus with the tyrosine hydroxylase-immunopositive fibers is the topic of an accompanying paper. Sparse tyrosine hydroxylase-immunopositive neurons were first observed in the anlage of the hypothalamus and septal region on the 13th fetal day. Their number increased progressively with age and by the 15th fetal day they already gave rise to a large dorsal accumulation. From the 18th fetal day on, tyrosine hydroxylase immunopositive neurons began to occupy their definitive positions, mainly concentrating within the hypothalamus: in the zona incerta, periventricular and arcuate nuclei. To a lesser extent, they were concentrated in the medial preoptic area, suprachiasmatic, supraoptic, paraventricular, dorsomedial, and anterior hypothalamic nuclei. The data on the distribution of the tyrosine hydroxylase-immunopositive neurons both in the hypothalamus and in the septal region during ontogenesis are summarized in the precise atlas. Primarily small bi- and unipolar catecholaminergic neurons first observed in the youngest fetuses undergo cytodifferentiation during ontogenesis, giving rise to at least two different populations localized ventrally, mainly in the arcuate nucleus, and dorsally, in the zona incerta. The neurons of the former population remain similar to those of the youngest fetuses, whereas the neurons of the latter increase significantly in size, forming several long, highly ramified processes.

Ontogenesis of tyrosine hydroxylase-immunopositive structures in the rat hypothalamus. Fiber pathways and terminal fields.

The innervation of the hypothalamus and septal region by catecholaminergic fibers was studied in rats from the 12th fetal day until the 9th postnatal day. Catecholaminergic fibers were visualized with preembedding immunocytochemistry using antibodies to tyrosine hydroxylase. An intensification of diaminobenzidine product with silver and gold was additionally applied to increase the sensitivity and resolution power of the routine immunocytochemical technique. It has been demonstrated that, from the 13th fetal day, the hypothalamus and the septal region receive catecholaminergic fibers either belonging to the hypothalamic neurons or coming with the medial forebrain bundle from the outside of the hypothalamus. As the development of the hypothalamus proceeds, these fibers form the extensive networks within some neurosecretory centers either containing (the zona incerta, periventricular nucleus, etc.) or almost lacking (suprachiasmatic and paraventricular nuclei) the catecholaminergic neurons. In the former case, they terminate on the processes or perikarya of catecholaminergic neurons, while in the latter case their varicosities surround the immunonegative presumptive neurons in a basket-like manner. Moreover, from the 18th fetal day catecholaminergic fibers penetrate between the ependymal cells towards the 3rd ventricle and the primary capillary plexus of the hypophysial portal circulation, apparently providing the release of catecholamines to the cerebrospinal fluid and portal blood, respectively. The data obtained in this study are considered as the morphological basis for the involvement of the hypothalamic catecholamines in neuroendocrine regulations during ontogenesis.

Development of the hypothalamic serotoninergic system during ontogenesis in rats. Immunocytochemical and radioautographic study.

The development of the hypothalamic serotoninergic system has been studied in rats from the 14th fetal till the 9th postnatal day. Serotoninergic elements were detected with immunocytochemistry using antiserum to serotonin (5-HT) and radioautography following the injections of [3H]5-HT into the cerebral ventricles. Immunocytochemistry failed to recognize hypothalamic 5-HT neurons either in fetuses or in neonatal rats. Conversely, radioautography demonstrated sparse radioactively labeled cells, undifferentiated in appearance, at the 16th and 18th fetal days. Moreover, at the 18th fetal day an accumulation of radioactively labeled typical neurons appeared in the suprachiasmatic region. By the 9th postnatal day, practically all radioactively labeled cells looking like highly differentiated neurons were concentrated in the dorsomedial nucleus. As to nerve fibers, a small number of 5-HT-immunoreactive axons arising from the raphe nucleus were first detected in the hypothalamus at the 16th fetal day. Two days later, both 5-HT-immunoreactive and radioactively labeled fibers became widely distributed through the hypothalamus with especially high concentrations in and around the optic chiasma. By the 9th postnatal day, the frequency of 5-HT fibers increased considerably both in the anterior and middle hypothalamus. Some 5-HT fibers were apposed to unlabeled neurons and others abutted on the capillaries either in the organum vasculosum of the lamina terminalis or in the median eminence. The present morphologic study suggests that during the perinatal period the hypothalamic 5-HT system becomes developed enough to be involved in the regulation of some neuroendocrine functions.

[Changes in testosterone levels in rabbit embryo testes after removal of the hypothalamus (encephalectomy)]. / Izmenenie kontsentratsii testosterona v semennikakh plodov krolika posle udaleniia gipotalamusa (éntsefaléktomii).

Brain hypothalamus region was removed by encephalectomy in utero in 21-23-day-old rabbit fetuses to find out when hypothalamic control over testicular androgenous function was set up during rabbit prenatal development. Testosterone concentration in testicular tissue was measured by radioimmunoassay at different terms after the operation. Encephalectomy resulted in a reliable decrease of testosterone concentration in 29- and 25-day-old fetuses, however, no significant changes were observed in 23-day-old fetuses. The greatest reduction in hormone gland concentration was noted in 25-day-old fetuses, encephalectomized on day 23 of their development. Introduction of LH-RH to 25-day-old encephalectomized fetuses 30 min before fixation significantly increased androgen concentration in the gland. The data obtained indicate that hypothalamic control over testicular androgenous function is set up on days 23-25 of prenatal development.

[Immunocytochemical and autoradiographic research on the growth of serotoninergic fibers to the cerebral ventricles in the perinatal period in rats]. / Immunotsitokhimicheskoe i avtoradiograficheskoe issledovaniia prorastaniia serotoninergicheskikh volokon k zheludochkam mozga v perinatal'nom periode u krys.

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.

Ependymal lining of infundibular recess in perinatal rats: relationships with portal capillaries and permeability.

Structure and permeability of the ependymal lining the infundibular recess were studied in perinatal rats with silver impregnation, electron microscopy, radioautography, and tracer techniques. According to our data basal processes of ependymal cells reach the primary portal plexus linking the 3rd ventricle and the hypophysial portal system all through the perinatal period. After birth, some of the processes penetrate into the perivascular space of the primary portal plexus and abut there on the endothelium of capillaries. Ependymal cells of fetuses and neonates are joined by specialized junctions (tight junctions, gap junctions and zonulae adhaerentes). Intraventricularly injected ionic lanthanum crosses the ependymal lining of fetuses both trans- and extra-cellularly everywhere in the infundibular recess. By postnatal day 9 only the rostral portion of the recess remains readily permeable. Caudally, extracellular leakage becomes highly restricted, apparently due to the appearance of circumferential tight junctions. Finally, [3H]dopamine seems to penetrate through the ependymal lining in the same way as ionic lanthanum entering the portal capillaries. These findings suggest that the adenohypophysiotropic neurohormones can penetrate from the cerebrospinal fluid into the portal circulation from the very beginning of the establishment of the hypothalamo-hypophysial functional relationships during ontogenesis.

Axovascular relationships in developing median eminence of perinatal rats with special reference to luteinizing hormone-releasing hormone projections.

Topographical relationships of neurosecretory axons with the capillaries of the primary portal plexus were studied in the median eminence of rats from the 14th fetal till the 9th postnatal day by means of electron microscopy combined with morphometric analysis. Special attention was given to the light and electron microscopic immunocytochemical examination of luteinizing hormone-releasing hormone projections to the median eminence. Neurosecretory axons possessing secretory granules and clear microvesicles were first observed in the median eminence at the 14th fetal day. However, all of them were situated at a distance from the primary portal plexus. By the 20th fetal day, neurosecretory axons reached the external basal lamina of the primary portal plexus giving rise to so-called axovascular contacts. Some axons even penetrated into the perivascular space, apparently facilitating the neurohormone delivery into the hypophysial portal circulation. From that time on, both the number of the axons abutting on the external basal lamina and the entire area of axovascular contacts increased gradually. As to luteinizing hormone-releasing hormone axons, they grew into the median eminence from the 18th fetal day concentrating in older fetuses and neonates either over the primary portal plexus, or around the infundibular recess of the 3rd ventricle. After birth, the concentration and distribution of luteinizing hormone-releasing hormone axons within the median eminence became similar to those of adults. Luteinizing hormone-releasing hormone axons were found to arise from the neurons of septopreoptic area including the diagonal band of Broca. These data suggest the onset of neurohormone release in the median eminence from the 14th fetal day followed by the establishment of the hypothalamic control over the pituitary functions during the perinatal period in rats.

Immunocytochemical and radioautographic study of serotonin projections to cerebral ventricles of perinatal 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 last case, 5-HT neuronal elements were detected according to their specific uptake of intraventricularly injected [3H]5-HT. At 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 toward its ventral portion. By the 9th postnatal day, extensive networks of supraependymal fibers became noticeable in the lateral ventricles and in the dorsal portion of the 3rd ventricle. In addition, a number of 5-HT fibers surrounded the infundibular and preoptic recesses and sometimes penetrated to the ventricular cavity. The functional significance of hypothalamic and ventricular 5-HT as a modulator of either the growth and differentiation of the developing brain or of some specific neuroendocrine functions is discussed.

[Changes in testosterone levels in the testes of rat fetus after removal of the hypothalamus]. / Izmenenie kontsentratsii testosterona v semennikakh plodov krys posle udaleniia gipotalamusa.

To specify the time of setting in of the hypothalamic control over testicular androgenous function in the prenatal development of rats, the brain hypothalamic area of 18.5-day-old fetuses was excised by encephalectomy in utero. Three days after the operation, i. e. on day 21.5 of the development, testosterone concentration in the testes was measured by radioimmunoassay. Testosterone concentration in the testes of encephalectomized fetuses was significantly decreased as compared with intact fetuses. Administration of LH-RH into the encephalectomized fetuses 2 hours prior to fixation removed the effect of encephalectomy on testosterone concentration in the testicular tissue. The data evidence the establishment of the hypothalamic control over androgenous function of the testes in rat fetuses at the end of the prenatal development.

Permeability of the blood-brain barrier in the median eminence during the perinatal period in rats.

An electron microscopic study has been carried out in order to examine the permeability of the blood-brain barrier in the median eminence of perinatal rats. After several minutes, intravascularly injected electron-dense tracers (lanthanum nitrate; horseradish peroxidase, 40000 MW1, ferritin, 500000 MW) pass the capillary wall, the perivascular space, and become incorporated into neurosecretory axons and basal processes of tanycytes both in fetuses and young rats. In the case of immature capillaries, the materials diffuse freely through the endothelial cells, and to a lesser extent are transferred via occasional plasmalemmal vesicles and fenestrae. As the maturation of capillaries proceeds, their permeability via plasmalemmal vesicles and fenestrae increases considerably due to a gradual rise of the number of these structures. The plasmalemmata of the differentiated endothelial cells become impermeable to all of the tracers. Only ionic lanthanum appears to penetrate through transendothelial channels and intercellular junctions between adjacent endothelial cells.

Light- and electron-microscopic study on the maturation of the primary portal plexus during the perinatal period in rats.

The maturation of the capillaries of the primary portal plexus in rats during the perinatal period has been studied light- and electron-microscopically. The number of capillaries covering the median eminence and of those invading the nervous tissue (capillary loops) increases significantly with age. Capillary loops were observed as early as the 18th fetal day. The mitotic divisions of the endothelial cells within the preexisting capillaries seem to be the main reason for the vascular growth. Immature capillaries with a characteristic narrow lumen are surrounded by a fuzzy basal lamina; their wall is formed by a generally expanded endothelium with rather sparse organelles and inclusions, and by minute flattened areas. The maturation of the capillary results in a progressive spread of flattened endothelium followed by an enlargment of the capillary lumen. Moreover, a rising concentration of organelles and inclusions, relatively numerous luminal microvilli, and a dense and uniform basal lamina become noticeable as capillary differentiation proceeds. These data are thought to reflect the progressive increase in the metabolic activity of the endothelium as well as the establishment of capillary patency during the perinatal period of rats.

Possible role of hypothalamus and hypophysis in the control of development of pancreas reactivity to the effect of glucose in rat fetuses.

To estimate the role of hypothalamus and hypophysis in the development of functional activity of pancreas, the changes of reactivity of pancreatic B-cells to glucose resulting from encephalectomy and decapitation of fetuses were investigated. Reactivity of pancreas was determined by the changes of insulin secretion induced by the addition of glucose into incubation medium. It was found that, when hypophysis and hypothalamus were removed as a result of decapitation of fetuses from normal or from diabetic pregnant rats on days 17.5-18.5 of development, their pancreas remained insensitive to glucose on 21.5 day. Removal of hypothalamus only when fetuses were encephalectomized on day 17.5 of development also resulted in the loss of sensitivity of fetal pancreas to glucose. Injection of hypothalamus homogenate to encephalectomized fetuses restored the stimulating effect of glucose on B-cells. When pancreas fragments of decapitated fetuses were preincubated together with adenohypophyses of adult rats, the decapitation effect was eliminated, and an increase of glucose concentration in the medium caused an intensive release of insulin. Similar restoring effect was induced by growth hormone (GH) and adrenocorticotrophin (ACTH) when used in in vivo and in vitro experiments. The data obtained give evidence of a possible contribution of hypothalamus and hypophysis to the control of the development of functional activity of pancreas in rat prenatal ontogenesis. However, the mechanism of this regulation remains unclear yet.

Studies on the hypothalamic control of thyroid function in the rabbit fetuses.

In order to reveal the onset in ontogenesis of hypothalamic control of thyroid function 22-23-day-old fetuses of rabbits were encephalectomized. The concentrations of T3 and T4 in thyroid glands and blood serum were measured radioimmunologically on the 29th and 30th day of prenatal life. Significant differences of T3 and T4 concentrations are revealed only in thyroid glands of 30-day-old encephalectomized fetuses. Treatment of encephalectomized fetuses by TRH 30 min prior to fixation removed the effect of encephalectomy on hormone concentration and on thyroid structure. The represented data are considered as a proof of the establishment of hypothalamic cotrol of hormone synthesis in thyroid gland beginning from the 30th day of prenatal life. The regulation of hormone release from the gland is probably established at a later stage of maturation.

[Role of the hypothalamus and pituitary gland in the development of pancreatic islet sensitivity to glucose in fetal rats]. / Znachenie gipotalamusa i gipofiza v razvitii chuvstvitel'nosti pankreaticheskikh ostrovkov ploda krysy k deistviiu gliukozy.

Differences in the pattern of the development of three enzymes of the plasma membrane have been established. The activity of Na, K-ATPase progressively increases, that of adenylate cyclase decreases, whereas the activity of 5-nucleotidase undergoes only slight changes during embryogenesis. Differences between these enzymes were also found with respect to the development of their sensitivity to the regulatory effects of catecholamines. Adrenaline reactivity of adenylate cyclase may be detected already in embryogenesis; it is lower than that in definite muscle tissue increasing during further ontogenesis. Catecholamine reactivity was not found in Na, K-ATPase and 5-nucleotidase up to the 17th day of incubation of chick embryos. The effect of adrenalin was observed at later stages of ontogenesis, it may be initiated by exogeneous cAMP and protein kinase. At postembryonic stages, similarity in the behavior of these enzymes was found with respect to the presence and pattern of their reaction to adrenalin (stimulation), as well as with respect to temporal dynamics of the effect. The data obtained indicate the existence of close connections between these enzymes, which are realized in the sequence adrenoreceptor-adenylate cyclase-cAMP-protein kinase-effector proteins.

[Effect of removal of the hypothalamus and pituitary gland from rat fetuses on the development of pancreatic B-cell function]. / Vliiani udaleniia gipotalamusa i gipofiza u plodov krysy na razvitie funktsii B-kletok podzheludochnoi zhelezy.

The reactivity of pancreatic B-cells of the decapitated and encephalectomized rat foetuses was determined by changes of insulin secretion in response to glucose introduction in the incubation medium. Upon removal of hypophysis together with hypothalamus in the intact rat females and females with alloxanic diabetes on the 17.5-18.5 day of prenatal development, their pancreas, unlike that of the control foetuses, remained refractory to glucose on the 21.5 day of development. The removal of hypothalamus only upon encephalectomy of the foetuses of the same age led to a similar effect, thus suggesting possible influence of hypothalamus on the regulation of functional maturation of the pancreatic B-cells in the end of prenatal development. Upon the combined incubation of pancreas fragments of the decapitated rat foetuses with adenohypophyses of the adult rats, the effect of decapitation was cut short and the gland responded by the increase of insulin secretion to the rise of glucose content in the medium.

[Changes in the concentration of triiodothyronine and thyroxine in rabbit fetuses following encephalectomy]. / Izmenenie kontsentratsii triiodotironina i tiroksina u plodov krolika posle éntsefaléktomii.

Triiodothyronine (T3) and thyroxin (T4) were detected by radioimmunoassay in the thyroid gland and serum of encephalectomized and intact rabbit fetuses. Encephalectomy performed on the 22-23d day of development did not produce any effect on thyroid hormone concentrations in 29-day-old fetuses. However, on the 30th day the concentrations of T2 and T4 in the thyroid gland were significantly lower in the encephalectomized fetuses than in the intact ones. These findings were consistent with changes in the morphological structure of the thyroid gland. The results obtained indicate that the hypothalamus begins to control thyroid function in the rabbit fetuses apparently from the 30th day of prenatal life.

Interrelations between neural elements and tanycytes during the perinatal period of the rat.

The present study has utilized a correlative scanning-transmission electron microscopic technique to examine interrelations between neural elements and differentiated tanycytes and to identify supraependymal cells in the ventral region of the 3rd ventricle during the perinatal period in the rat. From the 18th day of fetal life monoaminergic and/or peptidergic axons penetrate into the ventricle between the tanycytes. After birth, they form an extensive network covering the surface of the infundibular recess. The axons possess morphological characteristics suggestive of neurohormone secretion. From the 20th day of prenatal life subependymal axons begin to "innervate" the tanycytes. Supraependymal cells differ in their shape and ultrastructure, but all of them bear resemblance to macrophages, as they contain numerous lysosomes and phagosome-like bodies.

The adsorptive and transport capacity of tanycytes during the perinatal period of the rat.

Transport of ferritin and horseradish peroxidase from the 3rd ventricle to the median eminence was examined in rats during the perinatal life, the time when functional interrelations between hypothalamus and hypophysis are established. Protein tracers injected into the lateral ventricle are adsorbed on the apical surface of the tanycyte, mainly on its protrusions or in indentations. On the 18th day of prenatal life a few small bleblike protrusions are observed. After birth microvilli appear. In time their concentration increases to result in an increase of adsorbed substances. They are taken up by smooth and coated pinocytotic vesicles and transported to the basal portion of the cell or to the intercellular space bypassing junctional complexes. In addition to pinocytotic vesicles protein tracers fill channels of smooth ER or Golgi complex and multivesicular bodies illustrating a process probably involved in metabolic or secretory processes.