Adrenergic calcium signaling in astrocyte networks within the hippocampal slice

Abstract

Norepinephrine (NE) and glutamate (Glu) initiate intracellular calcium ([Ca2+](i)) transients, oscillations, and intracellular [Ca2+](i) waves in cultured astrocytes. To further elucidate the significance of NE- and Glu- evoked astrocytic [Ca2+](i), signaling to neuron-astrocyte communication in the mature CNS, [Ca2+](i), of astrocyte networks within hippocampal slices (P21-42) was measured during bath application of NE and Glu receptor agonists. Astrocytes in stratum radiatum were identified by highly negative membrane potentials (75 ± 3 mV), absence of action potentials, and dye coupling following intracellular injection of the [Ca2+]sensitive dye calcium orange. NE (2-100 μM) evoked [Ca2+](i), increases (7 of 8 slices, 24 of 24 cells in responding slices) characterized by an initial rise, 20-50 sec to peak, followed by a slower return to baseline (over ≃8 min). The α1-agonist phenylephrine (PE) (10-100 μM) evoked complex [Ca2+](i), signals (22 of 26 slices, 90 of 90 cells in responding slices) composed of both a prolonged component (5.1 ± 1.8 min), synchronized in neighboring cells, and multiple, mainly asynchronous [Ca2+](i) spikes (25.0 ± 11.6 sec). PE responses were completely blocked by the α1-antagonist prazosin (200 nM, n = 4 slices), but not by the α2-antagonist yohimbine (n = 3 slices). The α2-agonist clonidine (10-100 μM) did not increase [Ca2+](i) (n = 4 slices). α1-mediated [Ca2+](i) transients were observed after removal of extracellular [Ca2+](o) (n = 8 of 9 slices), indicating PE- induced Ca2+ release from intracellular stores. Adrenergic responses were mediated by α1-receptors localized to astrocytes because PE and NE increased [Ca2+](i) of acutely isolated hippocampal astrocytes. Glu (0.75- 2.0 nM) did not increase astrocytic [Ca2+](i) in slices (0 of 7), even in the presence of the Glu uptake inhibitor L-trans-pyrrollidine-2,4- dicarboxylic acid (PDC) (0 of 5 slices), or in acutely isolated astrocytes (0 of 7 cells). The metabotropic agonist t-ACPD (30 or 50 μM) did not increase astrocytic [Ca2+](i) in hippocampal slices (0 of 5), while kainate (200 μM or 1 mM) induced brief (1-2 min) [Ca2+](i) increases only rarely (2 of 8 applications in 6 slices). These results support a primary role of NE release and α1-adrenoceptor stimulation in neuron-astrocyte communication in the mature CNS.