Synaptically Released Acetylcholine Evokes Ca²⁺ Elevations in Astrocytes in Hippocampal Slices

Recent results have demonstrated the existence of bidirectional communication between glial cells and neurons. We investigated in brain slices whether rat hippocampal astrocytes respond to acetylcholine synaptically released by an extrinsic pathway. We stimulated the stratum oriens/alveus, which contains cholinergic afferents from the septum and diagonal band of Broca, and recorded whole-cell membrane currents and intracellular Ca²⁺ levels of astrocytes located in the hippocampal stratum oriens. Nerve-fiber stimulation evoked a long-lasting inward current and increased the Ca ²⁺ levels in astrocytes. Both astrocytic responses were abolished by tetrodotoxin or Cd²⁺ and were increased by 4-aminopyridine, indicating that the responses were attributable to synaptically released neurotransmitter. The inward current was inhibited by glutamate transporter antagonists, indicating that it was attributable to the electrogenic glutamate transporter activity. The synaptically evoked intracellular Ca²⁺ elevations were not affected by glutamate receptor antagonists but were abolished by atropine, indicating that they were mediated by muscarinic cholinergic receptors. Thapsigargin prevented the Ca²⁺ elevation but did not modify the inward current, indicating that the Ca²⁺ signal was attributable to intracellular Ca²⁺ mobilization. These results indicate that hippocampal astrocytes respond to acetylcholine released by synaptic terminals. The synaptically released acetylcholine acts on muscarinic receptors, mobilizing Ca²⁺ from the intracellular stores. Different regions in the recorded astrocytes showed independent stimulus-induced Ca ²⁺ variations, suggesting the existence of subcellular domains in the astrocytic responses evoked by the synaptic cholinergic activity. Therefore, our results show the existence of cholinergic neuron-astrocyte signaling and suggest that astrocytes are a target of axonal inputs from different brain areas.