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Journal of Neuroscience, Vol 16, 71-81, Copyright © 1996 by Society for Neuroscience

ARTICLE

In vitro ischemia promotes calcium influx and intracellular calcium release in hippocampal astrocytes

S Duffy and BA MacVicar
Department of Neuroscience, University of Calgary, Alberta, Canada.

The intracellular calcium concentration ([Ca2+])i of astrocytes within rat hippocampal slices was measured during simultaneous hypoxia and hypoglycemia to examine the early intracellular signaling events induced by this in vitro model of ischemia. Hypoxia-hypoglycemia for 3.3-7.5 min evoked [Ca2+]i increases in astrocytes iontophoretically loaded 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 absence of extracellular calcium ([Ca2+]o) (4/4 slices), indicative of Ca2+ release from internal stores. Hypoxia-hypoglycemia depolarized astrocytes (51 +/- 16 mV), suggesting additional contribution from voltage-gated Ca2+ influx. Depolarization of a similar magnitude (51 +/- 4 mV) by 50 mM extracellular potassium ([K+]o triggered [Ca2+]i increases (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 accurately isolated 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 R340/380 after 10 min) that were blocked by [Ca2+]o removal. Voltage- gated Ca2+ influx was still functional under ischemia, however, as 50 mM [K+]o evoked [Ca2+]i increases (14/14 cells, delta R340/380 of 48%) approximately equal to preischemic responses. Isolated neurons displayed large irreversible increases in basal [Ca2+]i after 1.5-6.5 min in vitro ischemia (10/12 cells; average delta R 340/380 of 152%). The absence of significant basal [Ca2+]i increases on isolated astrocytes 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.

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