In situ hybridization with digoxigenin labeled oligonucleotide probes: detection of CAMK-II gene expression in primary cultures of cerebral endothelial cells.

A better understanding of the regulation of gene expression under physiological and experimental conditions is one of the most important goals of today's neurobiology. In order to accomplish this task a number of in-situ hybridization methods have been elaborated. In the past few years the use of nonradioactive procedures have gained more and more space among these efforts. One of the most promising methods in this field is the application of digoxigenin-labeled probes, which have been used successfully in several laboratories. Here we present a rapid method providing good spatial resolution and low background labeling for the detection of messenger RNAs in various cell culture systems using digoxigenin-labeled probes. By using oligonucleotides complementary to the alpha and beta subunit of the calcium/calmodulin dependent protein kinase II (CAMK-II) we were able to demonstrate the presence of this enzyme in cultured cerebral endothelial cells, an enzyme that plays an important role in mediating the effects of extracellular signals.

Factors influencing astrocyte growth and development in defined media.

A previously described serum-free, defined medium (G2 medium) containing transferrin, selenium, hydrocortisone, biotin, fibroblast growth factor (FGF) and fibronectin developed for the growth of human and rat derived glioma cells was investigated for its ability to support proliferation of astrocytes in primary cultures of neonatal rat cerebrum. These cells were able to grow in G2 medium. Enhanced proliferation and repeated subcultivation were obtained after adding insulin and/or epidermal growth factor (EGF) to the G2 medium at concentrations of 5 µg/ml and 10 ng/ml, respectively. In these modified media (called G4 and G5 medium) astrocytes showed a higher degree of morphological differentiation as compared to serum supplemented medium. Cell type specificity was determined by immunocytochemical staining of glial fibrillary acidic (GFA) protein, which could already be demonstrated 5 days after plating cells. G4 and G5 represent the first serum-free defined media in which astrocytes proliferate and differentiate without preceding or intermediate contact to serum supplemented medium. Modification of the culture substratum by adding hyaluronic acid and chondroitin sulfate A to G4 medium (G2 medium + insulin) enhanced proliferation of astroglial cells by a factor of about 1.5. In the presence of epidermal growth factor no response to the altered culture dish surface was observed and the addition of fibronectin, otherwise a stringent plating requirement, was no longer necessary.

Retinal afferents form Gray-type-I and type-II synapses in the suprachiasmatic nucleus (rat).

The synapses of optic nerve afferents in the suprachiasmatic nucleus (SCN) usually form Gray type I (asymmetrical) synapses, but about 13% show clear-cut Gray type II ( symmetrical) active zones. Some presynaptic elements form a Gray type I--active zone with one dendrite and a Gray type II-active zone with another postsynaptic element at the same time. It is discussed whether this variabilility is related to a simultaneous excitatory and inhibitory action, to a variable activity or efficiency of the synapses or to various stages of maturation.

Self-innervation of dendrites in the rat suprachiasmatic nucleus.

Golgi-Cox impregnantions of the rat suprachiasmatic nucleus show small dendritic side branches which appear to contact their neurons of origin. Electronmicroscopically a dendrite has been found forming a Gray-type-II synapse with one of its own branches. The arrangement is discussed as a general phenomenon of a feedback connection for temporal limitation of local excitation.

Cerebrovascular ultrastructural alterations after intra-arterial infusions of ouabain, scilla-glycosides, heparin and histamine.

Cardiac glycosides which inhibit Na/K-ATPase (ouabain, scilliroside, scillirosidin) as well as heparin and histamine were infused into a cannulated branch of the middle cerebral artery or by isolated head perfusion in cats and dogs. Ouabain permeating the blood-brain barrier (BBB) caused the same selective swelling of astrocytes and of certain presynaptic elements as after direct application to the brain tissue. The other cellular elements of brain tissue and the vascular endothelium did not react, although the latter was exposed to the highest drug concentrations (about 10-3 M ouabain). By the swelling about one third of the capillaries became more or less constricted accompanied by an increase in endothelial vesiculation and in the number of osmiophilic inclusions in all cells of the vascular wall and of the pericapillary tissue. Osmiophilic material resembling plasma proteins occured in widened intercellular clefts indicating an increased BBB permeability after survival times (40 min). In contrast to the capillaries some terminal vessels are dilated which may correspond to shunt vessels causing an inhomogeneous, even increased cerebral blood flow after ouabain. Scilliroside and scillirosidin cause essentially the same changes as ouabain, but of smaller intensity and extent. In the present study, neither histamine nor heparin caused any structural change of the vessels or brain tissue.