Glutamate pretreatment affects Ca²⁺ signaling in processes of astrocyte pairs

Abstract

Simultaneous somatic patch-pipette recording of a single astrocyte to evoke voltage-gated calcium currents, and Ca²⁺ imaging, were used to study the spatial and temporal profiles of depolarization-induced changes in intracellular C^a2+ ([Ca²⁺]i) in the processes of cultured rat cortical astrocytes existing as pairs. Transient Ca²⁺ changes locked to depolarization were observed as microdomains in the processes of the astrocyte pairs, and the responses were more pronounced in the adjoining astrocyte. Considering the functional significance of higher concentrations of glutamate observed in certain pathological conditions, Ca²⁺ transients were recorded following pretreatment of cells with glutamate (500 μm for 20 min). This showed distance-dependent incremental scaling and attenuation in the presence of the metabotropic glutamate receptor (mGluR) antagonist, -methyl(4-carboxy-phenyl) glycine (MCPG). Estimation of local Ca²⁺ diffusion coefficients in the astrocytic processes indicated higher values in the adjoining astrocyte of the glutamate pretreated group. Intracellular heparin introduced into the depolarized astrocyte did not affect the Ca²⁺ transients in the heparin-loaded astrocyte but attenuated the [Ca²⁺]i responses in the adjoining astrocyte, suggesting that inositol 1,4,5 triphosphate (IP₃) may be the transfer signal. The uncoupling agent, 1-octanol, attenuated the [Ca²⁺]i responses in both the control and glutamate pretreated astrocytes, indicating the role of gap junctional communication. Our studies indicate that individual astrocytes have distinct functional domains, and that the glutamate-induced alterations in Ca²⁺ signaling involve a sequence of intra- and intercellular steps in which phospholipase C (PLC), IP₃, internal Ca²⁺ stores, VGCC and gap junction channels appear to play an important role.