Immunohistochemical and electron microscopic study of the rat hypothalamic nuclei and cell clusters under various experimental conditions

Summary In untreated, pregnant and thirsting rats the neurosecretory hypothalamic areas were investigated by means of the immunoperoxidase technique in order to demonstrate vasopressin- and oxytocin containing elements at the light- and electron microscopic level. In addition, chromalum-hematoxylinphloxin (CHP) staining and conventional double staining of ultrathin sections were used. The areas investigated included the anterior and posterior supraoptic nuclei, the paraventricular nuclei, the numerous accessory cell clusters in the region between the tractus opticus and the third ventricle as well as the median eminence. In all nuclei and in the accessory cell clusters, the number of vasopressin-reactive neurons exceeds that of oxytocin-reactive neurons. Compared with the anterior supraoptic nucleus, the posterior supraoptic nucleus and the accessory cell clusters react more heavily to prolonged thirst. In the median eminence the neurosecretory axons display close contacts with the portal vessels not only in its lateral portion but in thirsting animals also around the mid-line. There the internal layer is broadened and vasopressin-positive tanycytic processes reach the external zone. Parasagittally, fine vasopressin-positive material can be traced from the internal layer to small deposits at the portal vessels. In long term thirsting animals the typical feature of swollen axons exhibits a characteristic distribution in the median eminence and renders a distinct positive reaction to anti-vasopressin. The release of peptide hormones from the perikarya and from the axons within the nuclei as well as the mode of release within the median eminence are discussed. The significance of the positive immunostaining of the ependymal tanycytes and of some perikarya of the suprachiasmatic nucleus must be reconsidered by further studies.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/1) and Stiftung VolkswagenwerkDedicated to Professor Berta Scharrer on the occasion of her 70th birthdayThe author wishes to express her special gratitude to Dr. L.A. Sternberger for supplying the peroxidaseantiper oxidase-complex and to Dr. H. Stein (Pathologisches Institut der Universität Kiel) for supplying Anti-IgG. The skilful technical assistance of Mrs. H. Prien and Mrs. H. Schöning is thankfully acknowledged     

Localization of basic fibroblast growth factor in a subpopulation of rat sensory neurons

Summary The distribution of basic fibroblast growth factor (bFGF)-immunoreactivity (IR) was studied in rat sensory and autonomic ganglia. In postnatal and adult sympathetic superior cervical ganglia and in adult parasympathetic otic ganglia no bFGF-staining was found. Postnatal and adult neural crest-and placode-derived sensory ganglia displayed intensive bFGF-IR in a neuronal subpopulation. This subpopulation was characterized by use of consecutive sections of adult dorsal root ganglia stained with antibodies against substance P, somatostatin, bombesin, and bFGF. Basic FGF was colocalized with the somatostatin/bombesin subpopulation but not with substance P.     

Neural cells in culture: models for the purification and the study of the effects of growth factors

Conclusion and Perspectives We have summarized several methodologies currently available for the detection and analysisof protein factors affecting neural cells. As more knowledge is gained concerning growthfactors and their influences on cells and as the demand for bioassays increases, methodologicalimprovements are likely to be aimed at 1) decreasing yet the assay time; 2) increasingsensitivity of the measurements of cell responses; 3) increasing the number of simultaneouscellular responses measurable. A new very promising technique involves the use of severalfluorochromes having different cellular localizations and the continuous measurement of theirevolution in living cells by fluorospectrophotometry.The cell-blot method can be expected to extend to fractions submitted to other forms ofchromatography (native electrophoresis, isoelectric focusing, agarose). Other modificationsmay allow the detection of antagonistic factors, by including a stimulatory factor in the blotculture medium and measuring the suppression of a cellular response by the blotted opposingactivity.     

Protein Phosphotyrosine in Mouse Brain: Developmental Changes and Regulation by Epidermal Growth Factor, Type I Insulin-Like Growth Factor, and Insulin

Using antiphosphotyrosine antibodies, we have investigated protein phosphorylation in mouse brain during development in intact animals and in reaggregated cerebral cultures. Under basal conditions, in vivo and in vitro, the levels of two main phosphoproteins, of Mr 120,000 and 180,000 (pp180), increased with development, reaching a maximum in the early postnatal period and decreasing thereafter. In adult forebrain, pp180 was still highly phosphorylated, but it was not detected in cerebellum or in peripheral tissues. In reaggregated cortical cultures, epidermal growth factor (EGF), type I insulin-like growth factor (IGF-I), and insulin enhanced protein tyrosine phosphorylation of several proteins, which were specific for EGF or IGF-I/insulin. In highly enriched neuronal or astrocytic monolayer cultures, some proteins phosphorylated in basal conditions, or in response to EGF and IGF-I, were found in both types of culture, whereas others appeared cell type specific. In addition, in each cell type, some proteins were phosphorylated under the action of both growth factors. These results indicate that tyrosine protein phosphorylation is maximal in mouse brain during development and is regulated by growth factors in neurons as well as in astrocytes.     

The Role of Competence in the Cranio-Caudal Segregation of the Central Nervous System

Left to right thirds of Triturus presumptive prosencephalon show identical developmental potencies after implantation in a neutral Ambystoma environment. Such equipotential grafts were excised from stages between late-gastrula and mid-neurula and implanted into the neural plate of an Ambystoma host at different cranio-caudal levels. Their regional differentiation was independent of the age of the host, but dependent upon the age of the donor material; the older the latter the smaller the portion of the graft which was transformed into more posterior neural structures. Full transformation occurred in stage 11/12 grafts, while pure prosencephalic differentiation took place in stage 16 grafts, demonstrating that the period of competence of the neurectoderm for transformation extends from stage 11/12 up to stage 16. Irrespective of the level of implantation all grafts older than stage 11/12 and younger than stage 16 showed an uninterrupted cranially-oriented regional differentiation. The medio-lateral extension of the transformation process is primarily determined by the temporal loss of competence of the implanted neurectoderm. A comparison of grafts implanted at different cranio-caudal levels showed that transformation is more pronounced the more caudal the level of implantation, so that another factor(s) than competence must also play a role in the regional segregation of the CNS.     

Distal tubular segments of the rabbit kidney after adaptation to altered Na- and K-intake

Summary The baso-lateral cell-membrane area in kidney tubules appears to be associated with the capacity for electrolyte transport; in the rabbit, it decreases from the distal convoluted tubule (DCT-cells) over the connecting tubule (CNT-cells) to the cortical collecting duct (principal cells).Adaptation to low Na-, high K-intake changes this pattern: CNT-cells at the beginning of the connecting tubule have the highest membrane area, which decreases along the segment, but remains two-fold higher than in controls. Principal cells have a four-fold higher membrane area than in controls. Simultaneous treatment with the antimineralocorticoid canrenoate-K inhibits the structural changes in CNT-cells only in end-portions of the connecting tubule and in principal cells.After prolonged high Na-, low K-intake DCT-cells display a two-fold higher membrane area than controls, while CNT-cells and principal cells are not affected. Simultaneous treatment with DOCA does not affect the DCT-cells but provokes a moderate increase in membrane area in CNT-cells, and a 5.5-fold increase in principal cells.The data provide evidence that DCT-, CNT- and principal cells are functionally different cell types. The baso-lateral cell-membrane area, associated with electrolyte-transport capacity, appears to be influenced in DCT-cells mainly by Na-intake, in CNT-cells mainly by K-intake and in part also by mineralocorticoids, and in principal cells mainly by mineralocorticoids.Supported by SFB90, CARVAS, Heidelberg and Swiss National Science Foundation, Grant No. 3.900-0.79     

Mouse DNA sequences complementary to small nuclear RNA U1.

A mouse genomic library was screened for sequences complementary to U1 nuclear RNA. Out of the eight clones tested, none contained more than one copy of U1. Six of them were identical and one of those (clone 0U1-XIII) was further analyzed. This latter clone contained no other gene for discrete species of small size RNA in the 8 Kb EcoRI fragment encoding U1. A 248 bp Bg1II fragment from 0U1-XIII encompassing the full length of U1 as well as flanking regions on both sides has been subcloned and sequenced in M13 phage. Although the coding region was 96.5% homologous to rat U1a RNA, there is no direct evidence that this clone is a true gene. 3' and 5' flanking sequences of this as well as other published clones have been searched for homologies and the results of this search are discussed.     

Cloning and characterization of a genomic DNA fragment carrying the basic copy of the gene coding for variant surface antigen 118 of Trypanosoma brucei

It has been proposed (Hoeijmakers et al., 1980b) that variant surface antigen (VSA) gene expression in Trypanosoma brucei is accomplished by a gene re-arrangement involving the basic copy of the VSA gene to give the so-called expression-linked copy (which is present only in the strain expressing that particular antigen). In this publication, the basic and expression-linked copies of the gene have been visualized by Southern blot analysis of nuclear DNA and shown to be located on HindIII fragments of 4.5 and 10-12 kb, respectively. In addition, several other bands of weaker hybridization are seen, probably representing evolutionary relatives. Using a shotgun approach, HindIII gene banks have been constructed and recombinants isolated which carry the 4.5-kb HindIII fragment containing the VSA118 gene basic copy. Several clones containing evolutionary relatives were also found. The 4.5-kb HindIII fragment is able to hybridize to probes derived from both the 5' and 3' ends of the cDNA, while the relatives have homology only to the 3' end. A detailed comparison of the restriction map of VSA118 cDNA with that of the VSA118 basic copy showed no differences, demonstrating that the gene contains no introns. This result also indicates that the gene from which VSA118 mRNA is transcribed (whether this be the basic copy or the expression-linked copy) is identical to the basic copy over the region analysed.