Transfer of radioactive material between electrically coupled neurons of the leech central nervous system.

Intracellular application of tritiated precursors by means of microiontophoresis was performed on nerve cells in isolated segmental ganglia of the leech ventral nerve cord. Incorporation as well as intra- and interneuronal transport were studied by autoradiography after injection of fucose, glucosamine, glycine, leucine, orotic acid and uridine. With several minutes of intraneuronal injection the precursors were incorporated into macromolecules. Depending upon the tracer used, the radioactive material was distributed in a specific pattern over the cell somata and then released into the nerve processes. After application of orotic acid and uridine a transport of radioactive material, presumably RNA, could be observed in the processes of the injected neurons at a distance of about 200-500 mum. Fucose and glucosamine injection resulted in the most extended labeling of the nerve cell processes, indicating a transport rate of about 11 mm/day. When the radiochemicals were injected into one of the two electrically coupled giant nerve cells -- the so-called Retzius cells (Rc) -- a specific labeling not only of the injected Rc but also of the coupled but not injected Rc was found. Injection of protein or glycoprotein precursors into one Rc produced heavy labeling of both Rcs including their processes; a slight labeling of other ganglion compartments was only found after increasing the dosage of the amino acids glycine and leucine. With orotic acid and uridine this interneuronal transfer was confined to the electrically coupled Rc twin. Intracellular injection of one Rc with puromycin followed by injection of amino acids or fucose into the same Rc or into the coupled Rc resulted in an inhibition of precursor incorporation within the puromycin-injected Rc and an exclusive labeling of the coupled Rc, thus indicating that the precursors themselves were transferred. It is suggested that after microiontophoretic application an interneuronal transfer of relatively low molecular weight material takes place, probably across the low-resistance junction through which the Rcs are electrically coupled.

Microglia and microglia-derived brain macrophages in culture: generation from axotomized rat facial nuclei, identification and characterization in vitro.

In order to study microglial cells and microglia-derived brain macrophages in vitro, a method has been developed which allows the transfer of mitotic microglial cells from adult rat brain into tissue culture. The studies were performed on facial motor nuclei which were explanted after axotomy of the facial nerve. Outgrowing cells were identified and characterized by (i) morphological criteria using light and electron microscopy, (ii) in vivo [3H]thymidine labeling combined with subsequent in vitro autoradiography, (iii) immunocytochemistry for vimentin, GFAP, Fc and complement receptors, MHC antigens, laminin, fibronectin, factor VIII related- and 04 antigen as well as lectin histochemistry, and (iv) functional in vitro tests. In addition, a microglial cell line was established from proliferating cells. The results indicate that perineuronal microglia rather than astrocytes, perivascular cells, oligodendrocytes or endothelial cells may become phagocytic after having been activated by axotomy in situ.

Long-term cell culture of electrically active sensory neurons free of glial cells.

A method has been devised for long-term culturing of sensory ganglion cells, free of non-neuronal cells. After enzymatic-mechanical disaggregation of embryonic chick sensory ganglia the cell suspension is plated in dispersed cell culture. After 2-3 weeks in culture neurons and satellite cells re-associate in a histiotypic fashion. Partial or complete elimination of non-neuronal cells is achieved by treatment of the cultures with fluorodeoxyuridine. The glia-deprived neurons are electrophysiologically active and can be maintained in cell culture for more than 5 months.

Definition of a cell clone with astroglial characteristics derived from a chemically induced rabbit brain glioma.

Three-month-old rabbits were started on a fortnightly schedule of intravenous injections of N-methyl-N-nitrosourea. All but two of the central nervous system tumors induced in this manner were propagated in culture as permanent cell lines. On the 76RB-G-414-H line established from a grade 2 astrocytoma of this series of neoplasms, a cloning procedure was carried out using a laser microbeam. The clonal line originated in this way has been maintained in long-term culture and given the 76/RB-G-414-H-C designation. The cells of the clone display invariably a bipolar or multipolar configuration with long processes. Intermediate filaments are common and even abundant in some cells. Positivity for S-100 and GFA proteins is a regular finding in these cells. In addition, dibutyryl cyclic adenosine monophosphate treatment reduces cell division and stimulates cell process formation of these cells. Thus, it appears that we succeeded in establishing in vitro and maintaining in long-term culture a clone of tumor cells with astrocytic characteristics.

Induction of fiber outgrowth in PC12 pheochromocytoma cells by a neuronotrophic factor occurring in human tumors.

Human tumors contain one or several factors which induce fiber outgrowth in a clonal cell line of rat adrenal medullary pheochromocytoma. Its action was tested semiquantitatively in cultured PC12 pheochromocytoma cells using conditioned media from cultures of 45 different brain tumors and homogenates of 18 human tumors of brain and non-brain origin. All tumor homogenates investigated and 71% of the conditioned media were active. The stimulation of fiber growth and the morphological appearance of neurites resembled that seen after treatment with the nerve growth factor (NGF). However, experiments with NGF antiserum and with an NGF non-responsive PC12-mutant indicated that the neuronotrophic factor(s) occurring in human tumors are different from NGF. The active factor is heat labile, unstable at pH's below 4, but stable up to pH9. It retains its activity after dialysis.