The following two processes related to astrocytes are thought to depend on intercellular coupling through gap junctions: the spatial buffering of K+o and the spread of calcium waves in the astrocytic syncytium. We have used the following two independent methods to measure the open state of gap junctions: injection of lucifer yellow, and optical calcium imaging of calcium waves in response to probing the cells with a micropipette. The spread of lucifer yellow and calcium waves was inhibited if the cells were treated with either phorbol 12-myristate 13-acetate (PMA) or a synthetic diacylglycerol that activates protein kinase C. Down-regulation of protein kinase C by a 24-h treatment with PMA inhibited the uncoupling effect of PMA, supporting a direct involvement of protein kinase C in the regulation of astroglial gap junctions. Purinergic P2Y receptors, which are coupled to the inositol phospholipid pathway, are expressed by most astroglia in culture. Activation of the P2Y purinergic receptor with the selective agonist 2-methylthio-ATP uncoupled astroglia in a manner similar to the effect of treatment with PMA. Modulation of gap junctional conductance could isolate specific pathways within the astrocytic syncytium to form an extraneuronal information transfer network in brain.