Ultrastructural radioautographic analysis of neurogenesis in the hypothalamus of the adult frog, Rana temporaria, with special reference to physiological regeneration of the preoptic nucleus. I. Ventricular zone cell proliferation.

The localization and fine structure of proliferating cells in the hypothalamic preoptic area were studied by light- and electron-microscopic radioautography 1-2 h following single application of 3H-thymidine to adult Rana temporaria taken from their natural habitat in the spring and autumn. 3H-thymidine uptake by proliferating cells was much more pronounced in frogs caught in May/June, i.e., a month after the breeding period (labeled cells represent about 10% of the total ventricular zone cell population), compared to animals caught in mid-September, when it was very low. In both 3H-thymidine treatment groups the vast majority of labeled cells are found exclusively within the preoptic recess ventricular zone. With regard to ultrastructure, it contained proliferating cells of at least 4 types, ranging from immature forms (bipolar stem cells) to more differentiated elements (tanycyte-like ependymoblasts, "classical" ependymoblasts). All of them showed label over their nuclei indicating that these cells are capable of DNA synthesis and mitosis. The possible role of the preoptic recess ventricular zone as a source of precursor cells for new peptidergic neurosecretory cells, conventional neurons and glial cells in the hypothalamic preoptic area of the adult frog is discussed.

Ultrastructural radioautographic analysis of neurogenesis in the hypothalamus of the adult frog, Rana temporaria, with special reference to physiological regeneration of the preoptic nucleus. II. Types of neuronal cells produced.

Light- and electron-microscopic radioautography was used to identify the newly formed neuronal cells in the hypothalamic preoptic area of the frog. Adult Rana temporaria that had been caught in May/June received repeated 3H-thymidine injections and were sacrificed 30 days later. Heavily labeled cells were found in 1-micron plastic coronal sections of the preoptic area and then analysed in electron-microscopic radioautographs of neighbouring thin sections. The cells were identified as newly generated by the presence of 3H-thymidine label over the nucleus. All frogs showed considerable numbers of new peptidergic neurosecretory cells, small conventional neurons, and glia in the preoptic area. Some new ependymally located cells contacting the cerebrospinal fluid displaying ultrastructural characteristics of monoaminergic cells were also revealed. We conclude that prominent ventricular neurogenesis normally exists in the intact adult frog hypothalamus. The birth of small hypothalamic neurons seems to represent a case of sustained growth leading to a net increase in neuron numbers without loss. Conversely, the birth of large peptidergic neurosecretory cells, in which the increased secretory activity often leads to natural death of some cells, is considered as a neuronal replacement phenomenon, referred to as physiological regeneration of the magnocellular preoptic nucleus. The possible significance of this phenomenon in adult Anamnia is discussed.

[A method for preparing a monolayer of frog brain cells for the cytophotometric determination of their DNA content]. / Metod prigotovleniia monosloia kletok golovnogo mozga liagushki dlia tsitofotometricheskogo opredeleniia v nikh soderzhaniia DNK.

An improved method for the squashing of the brain cells permitting a good preservation of cell nuclei and their subsequent quantitative cytophotometric evaluation is presented. By this method a monolayer of the brain cells can be easily obtained. The method was tested in cytophotometric study of the DNA content in the Feulgen stained cells of the frog hypothalamic preoptic area. The proposed method can be used for the quantitative evaluation of different histochemical reactions in the brain cells.

[The characteristics of the hormonal profile of the central links in the regulation of adrenocortical function in rat postnatal ontogeny]. / Kharakteristika gormonal'nogo profilia tsentral'nykh zven'ev reguliatsii adrenokortikal'noi funktsii v postnatal'nom ontogeneze krysiat.

Radioimmune assay has been made of the ACTH content of the blood and adenohypophysis, as well as the content of vasopressin in the blood, median eminence, the posterior and intermediate hypophysis in 1, 3, 5, 7, and 20 days old rats and adult male rats. Comparison of the hormonal level in neurohaemal areas with that in the blood plasma suggests that transadenohypophyseal path of hypothalamic neurohormonal control of the adrenocortical function becomes essentially important only after formation of capillary plexus in the external zone of the median eminence.

[The hormonal indices of the reactivity of the central links in the hypothalamo-hypophyseal-adrenocortical system of rats during postnatal ontogeny]. / Gormonal'nye pokazateli reaktivnosti tsentral'nykh zven'ev gipotalamo-gipofizarno-adrenokortikal'noi sistemy (GGAKS) krys v khode postnatal'nogo ontogeneza.

Radioimmunological assay has been made of the amount of ACTH in the adenohypophysis and blood plasma in adult male rats as well as in 1, 3, 5, 7, and 20 days old rat puppies 30 minutes after a surgical stress, i. e. cutting the skin at the back of the body. Reliable increase of ACTH level was originally observed in 7-day puppies, whereas in younger animals surgical stress resulted in the decrease of ACTH in the blood. Changes in vasopressin content of the blood and hypophysis exhibit significant variations, but on the whole mainly the decrease was observed in younger animals together with the increase from the 5-7th day of postnatal development of rats. Accumulation of vasopressin was noted in the median eminence after surgical stress in younger animals. The data obtained suggest that the early period of development of the hypothalamo-hypophyseal-adrenocortical system is characterized rather by paradoxal reaction than by non-reactivity, which may be associated with poor neurohormonal transport in the outer zone of the median eminence.

A cytophotometric analysis of the structure of hypothalamic cell populations in the frog, Rana temporaria (L.), with special reference to seasonal changes in chromatin status.

We used cytophotometry after the Feulgen reaction and UV cytophotometry to measure the DNA content of quiescent cells of the hypothalamic preoptic region (HPR) of adult and juvenile frogs (Rana temporaria) that had been caught in their natural habitat in winter, spring and summer. The histone-to-DNA ratio in cell nuclei was cytophotometrically determined using a combined Feulgen, heparine and alcian-blue staining procedure. The vast majority of HPR cells studied had nuclei with a diploid DNA content. However, we observed great variability in the Feulgen-DNA content of the HPR cell population, which was not detected in the diploid standard (hepatocytes). This heterogeneity in the diploid sample of the HPR cell populations was always greater in prespawning frogs and may have been due to differences in the chromatin arrangement in nuclei. About 1% of cells had a DNA content either ranging between diploid and tetraploid levels (H2C cells) or at the tetraploid level (4C and 2C x 2 cells). The proportion of these cells was not affected by the age of the animals or the annual cycle, thus suggesting that there is no age-related increase in the mean DNA content in the frog HPR. The mean DNA contents of H2C and 4C cells were much higher than those in the standard (hepatocytes). This cannot be simply attributed to the presence of different amounts of nuclear proteins, but rather indicates that at least a certain proportion of the highest DNA contents may be due to a real extra-DNA synthesis.

[Cytophotometric analysis of the cell population composition of the the preoptic area of the hypothalamus in the common frog]. / Tsitofotometricheskii analiz sostava kletochnoi populiatsii preopticheskoi oblasti gipotalamusa travnoi liagushki.

DNA contents in squashed cells of the adult frog hypothalamic preoptic region (HPR) were measured using the Feulgen and UV cytophotometry techniques. The histone-DNA ratio in the cell nucleus was determined by means of a combined Feulgen-heparin-Alcian blue staining procedure. The nuclei of the vast majority of HPR cells have a diploid DNA content. However, in cells of this group the mean values of DNA amount and the distribution range were always higher than those in hepatocytes used as a diploid standard. Such a heterogeneity in DNA content in the diploid part of HPR cell population could apparently suggest some differences in the nuclear chromatin arrangement to be always higher in spring before the frog spawning, and it seems to be characteristic of this type of cells. About 1 per cent of cells with hyperdiploid surplus of DNA (H2c cells) as well as of tetraploid cells (4c and 2c X 2 cells) is found in HPR in frogs sacrificed both in winter and in summer. The quota of these cells has no reference either to the frog's age or to the annual cycle. The fact that the mean DNA values in H2c and 4c cells are much higher than in the standard cannot be explained by the presence of different amounts of nuclear proteins only. It is suggested that at least some part of the highest DNA values may be due to an actual extra DNA synthesis in a small constantly existing pool of HPR cell population.

[Mitotic division of differentiated neurosecretory cells in the hypothalamus of adult amphibians]. / O mitoticheskom delenii differentsirovannykh neirosekretornykh kletok gipotalamusa vzroslykh amfibii.

By means of multiple 3H-thymidine administration to adult frogs, a direct electron microscopic radioautographic evidence is first by presented regarding the possibility of an in vivo mitotic division of differentiated hypothalamic peptidergic neurosecretory cells. The absence of any signs of cytoplasmic dedifferentiation, as well as a poor development of spindle microtubules in this cell may be suggestive of a polyploidizing, possibly acytokinetic mitotic process occurring in such kind of cells.

[Cytophotometric study of the DNA content in the cells of the preoptic area of the hypothalamus in hibernating common frogs]. / Tsitofotometricheskoe issledovanie soderzhaniia DNK v kletkakh preopticheskoi oblasti gipotalamusa u zimuiushchikh travianykh liagushek.

The DNA content in the squashed Feulgen stained cells of the hypothalamic preoptic region in wintering adult and juvenile frogs was measured cytophotometrically. The vast majority of hypothalamic cells studied showed diploid DNA contents. Only 0.9% of tetraploid cells (4c and 2c X 2 cells) was found in this region, both in adult and juvenile frogs. The incidence of tetraploid hypothalamic cells varied among the individuals studied. In some adult frogs as well as in some juveniles no tetraploid cells were detected. The data obtained do not confirm the age related increase in the mean DNA content in frog's hypothalamic cells population.

[Autoradiographic study of the proliferative activity of the preoptic recess ependyma in yearling and juvenile common frogs]. / Avtoradiograficheskoe issledovanie proliferativnoi aktivnosti épendimy preopticheskoi bukhty u odnoletnikh i dvukhletnikh osobei travianoi liagushki.

Proliferative activity of the ependyma, lining the recessus praeopticus in juvenile frogs was studied with 3H-thymidine radioautography. Usually much more pronounced proliferation of ependymal cells occurred in the preoptic region in one year old frogs as compared with two year old ones. It can be concluded that in the former animals the migration of postmitotic labeled cells into the subependymal zone of the recessus preoptic area is significantly more intense. By the 30th day after multiple isotope injections some newly formed neurosecretory cells with labeled nuclei were found in the 1-5 cellular position of the recessus praeopticus subependymal zone. It is postulated that in juvenile frogs the ependyma of lateral wall of recessus praeopticus is probably a source ("cambium"), from which some young neurosecretory cells may originate.

The hypothalamo-hypophysial system of the lamprey, Lampetra fluviatilis L. III. High-resolution radioautography of monoaminergic structures in neurohemal regions.

The distribution of monoaminergic structures was studied in the proximal neurosecretory contact region and neurohypophysis of the lamprey by light and electron microscopic radioautography. Only weak radioautographic reactions were found in the proximal neurosecretory contact region 1 h after injection of 3H-dopamine. High-resolution radioautography revealed some labeled neurosecretory terminals mainly in contact with the basement membrane of the connective tissue layer separating the proximal neurosecretory contact region from the hypophysial pars distalis. The number of silver grains as well as the number of neurosecretory terminals marked by the presence of labeled dopamine was much higher in the neurohypophysis of the same species. In the latter, labeled neurosecretory terminals were found in contact with the connective tissue layer containing blood vessels of the general circulation. Some neurosecretory terminals make synaptoid contacts with tanycyte perikarya and their basal processes. According to their ultrastructure and the size of their granules, the labeled neurosecretory terminals are identical with the B type terminals described in both neurohemal regions (transmission electron microscopy). No labeled neurosecretory terminals were observed in the proximal neurosecretory contact region and the neurohypophysis of lampreys treated with the serotonin precursor, 3H-5-hydroxytryptophan.

Quantative radioautographic light and electron microscopic analysis of the localization of monoamines in the median eminence of the rat.

Serotonin containing structures in the median eminence of the rat have been studied by quantitative light and electron microscopic radioautography following intraventricular infusion of tritiated 5-hydroxytryptophan. One hour after injection of the tracer the highest density of silver grains was recorded in the ependymal and external zones, especially in the lateral palisade zone. The proportion of labelled neurosecretory terminals was also larger in the lateral palisade zone (29%) as compared with the medial palisade zone (13%), although the mean number of developed silver grains per one terminal was higher in the latter. On the average, 16% of neurosecretory terminals sequestered radiolabelled 5-hydroxytryptophan in the external zone of the rat median eminence. It is suggested that serotonin, like catecholamines, is discharged from neurosecretory terminals localized in the external zone and via the portal circulation affects the function of the anterior pituitary. The sites of origin of serotoninergic structures of the median eminence as well as the possible role of monoamine (catecholamine and indolamine) neurohormones in a dual peptidergic and monoaminergic control of anterior pituitary functions are discussed.

Quantitative radioautographic light and electron microscopic analysis of the localization of monoamines in the median eminence of the rat. I. Catecholamines.

By means of light and electron microscopic radioautography a quantitative study of the regional distribution of catecholamines in the median eminence of the rat was carried out. One hour after intraventricular injection of 3H-dopamine the highest radioautographic reaction was recorded in the external zone, especially in the lateral palisade zone where many neurosecretory terminals are separated from the basal lamina of the portal pericapillary space by a glial "cuff". This area showed the highest percentage (52%) of labelled catecholamine containing neurosecretory terminals as well as the maximal silver grain density per one terminal. In the medial palisade zone where direct neurovascular contacts with the capillary loops prevail, only 27 per cent of neurosecretory terminals were found to harbour tritiated dopamine. On the average 35 per cent of neurosecretory terminals in the median eminence of the rat contain catecholamines (both dopamine and noradrenaline). Pretreatment of animals with recretory terminals as well as grain density over terminals were decreased in both the medial and lateral palisade zones, although to a lesser degree in the latter. The site of origin of catecholamine fibers as well as the mode of catecholamine action at the level of the median eminence are discussed.