HIV-1 GP120 GLYCOPROTEIN INDUCES [CA2+](I) RESPONSES NOT ONLY IN TYPE-2 BUT ALSO TYPE-1 ASTROCYTES AND OLIGODENDROCYTES OF THE RAT CEREBELLUM

Cultures of cerebellar cortex cells were exposed to the HIV-1 envelope glycoprotein, gp120, and investigated for cytosolic Ca2+ ion concentration ([Ca2+](i)) changes by the fura-2 ratio videoimaging technique while bathed in complete, Na+-free or Mg2+-free Krebs-Ringer media. At the end of the [Ca2+]i experiments the cells were fixed and immunoidentified through the revelation of markers specific for neurons (microtubule associated protein-2), type-2 (A2B5) or all (glial fibrillary acidic protein) astrocytes, oligodendrocytes (galactocerebroside) or microglia (F4/80 antibody). In complete medium, rapid biphasic (spike-plateau) responses induced by gp120 (0.1-1 nM) were observed in a subpopulation of type-2 astrocytes. In addition, slow but progressive responses were observed in other type-2 cells and oligodendrocytes, whereas type-1 astrocytes showed small responses, if any, and granule neurons did not respond at all. Use of Na+-free medium (a condition that blocked another gp120-induced response, cytosolic alkalinization) resulted in an increase in [Ca2+](i) response that was appreciable not only in type-2 but also in most type-1 astrocytes, possibly because of the inhibition of the Na+/Ca2+ exchanger and the ensuing decrease in Ca2+ extrusion. Granule neurons, including those in direct contact with responsive astrocytes, remained unresponsive, even when the experiments were carried out in Mg2+-free medium supplemented with glycine, a condition that favours activation of the glutamatergic N-methyl-D-aspartate (NMDA) receptor. The results obtained demonstrate that sensitivity to gp120 is a property of not only a few type-2 astrocytes but of the majority of cerebellar glial cells, which, however, do not respond to the protein with glutamate release, as indicated by the negative results obtained with NMDA-receptor-expressing granule neurons. Single glial cell [Ca2+](i) increase, the faster and most sensitive effect of gp120 revealed in the brain so far, could be ultimately employed to reveal CD4-independent transmembrane signalling machanisms of the viral protein that, at the moment, remain almost entirely unknown.