IN-VITRO ISCHEMIA PROMOTES CALCIUM INFLUX AND INTRACELLULAR CALCIUM-RELEASE IN HIPPOCAMPAL ASTROCYTES

The intracellular calcium concentration ([Ca2+](i)) of astrocytes with in rat hippocampal slices was measured during simultaneous hypoxia and hypoglycemia to examine the early intracellular signaling events indu ced by this in vitro model of ischemia. Hypoxia-hypoglycemia for 3.3-7 .5 min evoked [Ca2+](i) increases in astrocytes iontophoretically load ed with calcium orange (11/14 slices; 2.5 min to peak [Ca2+](i), 5 min to >60 min duration). Calcium elevations also were observed in the ab sence of extracellular calcium ([Ca2+](o)) (4/4 slices), indicative of Ca2+ release from internal stores, Hypoxia-hypoglycemia depolarized a strocytes (51 +/- 16 mV), suggesting additional contribution from volt age-gated Ca2+ influx. Depolarization of a similar magnitude (51 +/- 4 mV) by 50 mM extracellular potassium ([K+](o)) triggered [Ca2+](i) in creases (20/24 slices), which were blocked by removal of [Ca2+](o) (8/ 8 slices) indicating that depolarization promoted Ca2+ influx, Voltage -gated Ca2+ influx and internal release were measured in acutely isola ted astrocytes during in vitro ischemia to examine these processes in the absence of surrounding neurons, Hypoxia-hypoglycemia (7.5-34.0 min ) induced only modest, slow increases in the basal [Ca2+](i) of Fura-2 -loaded isolated astrocytes (average 12% increase in Fura-2 ratio R(34 0/380) after 10 min) that were blocked by [Ca2+](o) removal, Voltage-g ated Ca2+ influx was still functional under ischemia, however, as 50 m M [K+], evoked [Ca2+](i) increases (14/14 cells, Delta R(340/380) of 4 8%) approximately equal to preischemic responses. Isolated neurons dis played large irreversible increases in basal [Ca2+](i) after 1.5-6.5 m in in vitro ischemia (10/12 cells; average Delta R(340/380) of 152%). The absence of significant basal [Ca2+](i) increases in isolated astro cytes indicates that ischemia-induced Ca2+ influx and internal release in astrocytes within slices depend on signals released from neurons ( K+, neurotransmitters). Ischemic [Ca2+](i) elevations may constitute a signaling mechanism for postischemic reactive responses.