COLOCALIZATION OF LOW-AFFINITY AND HIGH-AFFINITY NGF RECEPTORS ON PC12 CELLS, C6 GLIOMA-CELLS AND DORSAL-ROOT GANGLION NEURONS

The biological responsiveness of neural cells to nerve growth factor (NGF) appears to require expression and ligand binding to both the low-affinity NGF receptor (LNGFR) and the proto-oncogene product trk, the latter being a receptor tyrosine kinase. Immunolocalization of the LNGFR and the high-affinity component of the NGF receptor, trk (HNGFR) was studied by electron microscopic morphometric analysis on cultured PC12 pheochromocytoma cells, C6 glioma cells and neonatal rat dorsal root ganglia neurons using a double immunogold labeling technique. Two receptor-specific antibodies, anti-LNGFR monoclonal antibody 192-IgG and a polyclonal antibody against the 14 carboxy-terminal amino acids of the Trk protein, were utilized in conjunction with immunoglobulin conjugated to colloidal gold particles of different sizes. All cells treated with NGF (50 ng/ml) displayed significant colocalization of LNGFR/HNGFR-like immunoreactivity. Gold particles associated with LNGFR (LNGFR-like immunoreactivity) were frequently seen near 2 or 3 (or more) particles delineating the HNGFR on all cell surfaces. Positive Trk-like immunoreactivity (HNGFR) thus seems to localize in close proximity to LNGFRs in at least these cell types

GLUCOCORTICOIDS DEPRESS ACTIVITY-DEPENDENT EXPRESSION OF BDNF MESSENGER-RNA IN HIPPOCAMPAL-NEURONS

Glucocorticoid hormones are important regulators of brain development and ageing, and can impair the capacity of hippocampal neurones to survive various neurological insults. Here we show that dexamethasone, a synthetic glucocorticoid, prevents activity-dependent increases of brain-derived neurotrophic factor (BDNF) mRNA in cultures of rat hippocampal neurones. In situ hybridization was used to evaluate the levels of BDNF mRNA. Up-regulation of BDNF mRNA triggered by depolarization with high potassium, or exposure to the glutamate receptor agonist kainic acid, resulted both from higher levels of expression in neurones and from new recruitment of cells. These data suggest that the known ability of glucocorticoids to exacerbate neuronal injury following ischaemia and other metabolic insults could be due to antagonism of regulatory mechanisms governing neurotrophin levels in the brain

INTERACTION OF GANGLIOSIDE GM1 WITH THE B-SUBUNIT OF CHOLERA-TOXIN MODULATES INTRACELLULAR FREE CALCIUM IN SENSORY NEURONS

The B subunit of cholera toxin, which binds specifically to GM1 ganglioside on cell surfaces, has previously been shown to modulate intracellular calcium levels and growth in several cell types. To explore a role for such changes in calcium in the growth regulatory function of cell-associated GM1 in neurons, dissociated neurons from chicken embryonic day 8 dorsal root ganglia were exposed to the B subunit. To enhance sensitivity to B subunit, some neurons were also enriched with added GM1 (100 microM) and then exposed to B subunit. Incubation of naive cultures with 1 microgram/ml of the B subunit was sufficient to produce modest increases in intracellular free calcium above basal levels in a minor percentage of cells for at least 5 min, as measured by fura-2 fluorescence imaging. Pretreatment of the cells with GM1 for 48 hr increased even further the elevations in intracellular free calcium and the percentage of responding neurons observed after B subunit exposure. These increases in intracellular calcium required the presence of external Ca2+, but were not inhibited by calcium channel blockers. Such changes in calcium were accompanied by fine alterations in morphology affecting mostly the branching of neurites and were more pronounced in the presence of GM1. However, the morphological changes did not result in altered neurofilament protein expression. Immunogold electron microscopy using anti-choleragenoid depicted extensive aggregations of immunoreactive gold particles on neuronal surfaces, which were more extensive in cells treated with GM1. The results demonstrate that cell incorporated GM1 may modulate calcium fluxes, perhaps accounting for the growth regulatory functions of GM1 in both neuronal and other cell types