NONLINEAR PROPAGATION OF AGONIST-INDUCED CYTOPLASMIC CALCIUM WAVES INSINGLE ASTROCYTES

In astrocytes in primary culture, activation of neurotransmitter recep tors results in intracellular calcium signals that propagate as waves across the cell. Similar agonist-induced calcium waves have been obser ved in astrocytes in organotypic cultures in response to synaptic acti vation. By using primary cultured astrocytes grown on glass coverslips , in conjunction with fluorescence microscopy we have analyzed agonist -induced Ca2+ wave initiation and propagation in individual cells. Bot h norepinephrine and glutamate elicited Ca2+ signals which were initia ted focally and discretely in one region of the cell, from where the s ignals spread as waves along the entire length of the cell. Analysis o f the wave propagation and the waveform revealed that the propagation was nonlinear with one or more focal loci in the cytoplasm where the w ave was regeneratively amplified. These individual loci appear as disc rete focal areas 7-15 mu m in diameter and having intrinsic oscillator y properties that differ from each other. The wave initiation locus an d the different amplification loci remained invariant in space during the course of the experiment and supported an identical spatiotemporal pattern of signalling in any given cell in response to multiple agoni st applications and when stimulated with different agonists which are coupled via InsP(3). Cytoplasmic Ca2+ concentration at rest was consis tently higher (17 +/- 4 nM, mean +/- S.E.M.) in the wave initiation lo cus compared with the rest of the cytoplasm. The nonlinear propagation results from significant changes in signal rise times, amplitudes, an d wave velocity in cellular regions of active loci. Analysis of serial slices across the cell revealed that the rise times and amplitudes of local signals were as much as three- to fourfold higher in the loci o f amplification. A phenomenon of hierarchy in local amplitudes of the signal in the amplification loci was observed with the wave initiation locus having the smallest and the most distal locus having the larges t amplitude. By this mechanism locally very high concentrations of Ca2 + are achieved in strategic locations in the cell in response to recep tor activation. While the average wave velocity calculated over the le ngth of the cell was 10-15 mu m/s, in the active loci rates as high as 40 mu m/s were measured. Wave velocity was fivefold lower in regions of the cell separating active loci. The differences in the intrinsic o scillatory periods give rise to local Ca2+ waves that show the propert ies of collision and annihilation. It is hypothesized that the wave fr ont provokes regenerative Ca2+ release from specialized areas in the c ell where the endoplasmic reticulum is endowed with higher density of InsP(3) receptor channels. Thus wave propagation is achieved by a proc ess of diffusion and regenerative Ca2+ release in multiple cellular lo ci provoked by the advancing wave front; in this way, wave propagation is nonlinear and saltatory. Regenerative Ca2+ wave propagation from d istal astrocytic processes to the cell body and neighboring cells is l ikely to provide an important signalling mechanism in the nervous syst em. (C) 1994 John Wiley and Sons, Inc.