Astrocytes posses a form of excitability based on intracellular Ca²⁺ variations. Using Ca²⁺ imaging and electrophysiological techniques in cultured rat hippocampal cells we determined the electrophysiological consequences of elevating astrocytic Ca²⁺ levels on neurons. Astrocytic Ca²⁺ elevations caused a slow inward current (SIC) in adjacent neurons that was mediated by ionotropic glutamate receptor activation. Glutamate (Glu) was released by astrocytes because the insensitivity of the SIC to tetanus toxin, which prevents neuronal exocytosis. Furthermore, thapsigargin, injection of BAPTA, and photolysis of Ca²⁺ cages demonstrated that a Ca²⁺ elevation in astrocytes is both necessary and sufficient to stimulate Glu release. To further investigate the mechanisms underlying the astrocytic Glu release we analysed the effects of disrupting astrocytic vesicle proteins. We found that Glu release requires an electrochemical gradient necessary for Glu uptake in vesicles, because bafilomycin A₁ reduced neuronal responses. Furthermore, neuronal responses were inhibited by injection of astrocytes with the light chain of Botulinum B that cleaves the SNARE protein synaptobrevin. These results demonstrate that the Ca²⁺-dependent Glu release from astrocytes is a SNARE protein-dependent process that requires the presence of functional vesicle-associated proteins, suggesting that astrocytes store Glu in vesicles and that it is released by exocytosis.

We also analysed the effects of astrocytic Ca²⁺ elevations on synaptic transmission. Ca²⁺ elevation in astrocytes increased the frequency of excitatory as well as inhibitory miniature postsynaptic currents (mPSCs), without modifying their amplitudes. This AP5-sensitive astrocytic-induced increase of mPSC frequency was due to activation of NMDA receptors, which are located extrasynaptically because it persisted after blockage of synaptic receptors by MK-801. Therefore, astrocytes modulate spontaneous synaptic transmission by increasing the probability of transmitter release through the activation of NMDA receptors. In addition, stimulation of astrocytes transiently reduced the magnitude of action potential-evoked excitatory and inhibitory postsynaptic currents through the activation of metabotropic Glu receptors.

These results demonstrate that astrocytes regulate neuronal excitability and synaptic transmission (for a review, see Araque et al. 2001).

Considering the relevance of Ca²⁺ elevations in astrocytes, our work has recently focused on the mechanisms involved in the astrocytic Ca²⁺ responses to synaptic activity. Hippocampal astrocytes respond with Ca²⁺ elevations to synaptically released Glu (Porter & McCarthy, 1996). We investigated in rat hippocampal slices whether astrocytes respond to a different synaptically released neurotransmitter by an extrinsic pathway. We stimulated the alveus, which contains glutamatergic axons as well as cholinergic afferents from the septum and diagonal band of Broca, and recorded currents and Ca²⁺ levels of astrocytes located in the stratum oriens. The stimulation evoked an inward current due to Glu transporter activity, and increased the Ca²⁺ levels in astrocytes. The responses were enhanced by 4-AP, and abolished by TTX or Cd²⁺, indicating that they were due to synaptically released neurotransmitter. Ca²⁺ variations were unaffected by Glu receptor antagonists, but were abolished by atropine, indicating that astrocytes respond to acetylcholine released by synaptic terminals. These results show the existence of cholinergic neuron-to-astrocyte signalling, and suggest that astrocytes are a target of axonal inputs from different brain areas.

Although stratum oriens astrocytes express Glu receptors, the alveus stimulation did not evoke Glu-mediated Ca²⁺ elevations. We investigated whether a different glutamatergic pathway could activate those receptors. We recorded astrocytic responses to the stimulation of glutamatergic Schaffer collaterals (SC). This stimulation induced an inward current mediated by Glu transporters, and evoked astrocytic Ca²⁺ elevations that were abolished by Glu receptor antagonists.

These results indicate that while stratum oriens astrocytes showed acetylcholine-mediated Ca²⁺ elevations after alveus stimulation, astrocytes responded with Ca²⁺ elevations to Glu released by SC. Therefore, astrocytes show functional sub-cellular domains and may discriminate between the activity of different synaptic terminals belonging to different axon pathways.

In conclusion, these results support the existence of a complex bidirectional communication between astrocytes and neurons, and indicate an important active role of astrocytes in the physiology of the nervous system.

This work was supported by Ministerio de Ciencia y Tecnología, Spain. G.P. is a predoctoral fellow from MCyT.