Astrocytes comprise approximately half of the volume of the adult mammalian brain and are the primary neuronal structural and trophic supportive elements. Astrocytes are organized into distinct nonoverlapping domains and extend elaborate and dense fine processes that interact intimately with synapses and cerebrovasculature. The recognition in the mid 1990s that astrocytes undergo elevations in intracellular calcium concentration following activation of G protein-coupled receptors by synaptically released neurotransmitters demonstrated not only that astrocytes display a form of excitability but also that astrocytes may be active participants in brain information processing. The roles that astrocytic calcium elevations play in neurophysiology and especially in modulation of neuronal activity have been intensely researched in recent years. This review will summarize the current understanding of the function of astrocytic calcium signaling in neurophysiological processes and discuss areas where the role of astrocytes remains controversial and will therefore benefit from further study.

The concept that astrocytes release neuroactive molecules (gliotransmitters) to affect synaptic transmission has been a paradigm shift in neuroscience research over the past decade. This concept suggests that astrocytes, together with pre- and postsynaptic neuronal elements, make up a functional synapse. Astrocyte release of gliotransmitters (for example, glutamate and adenosine triphosphate) is generally accepted to be a Ca2+-dependent process. We used two mouse lines to either selectively increase or obliterate astrocytic Gq G protein-coupled receptor Ca2+ signaling to further test the hypothesis that astrocytes release gliotransmitters in a Ca2+-dependent manner to affect synaptic transmission. Neither increasing nor obliterating astrocytic Ca2+ fluxes affects spontaneous and evoked excitatory synaptic transmission or synaptic plasticity. Our findings suggest that, at least in the hippocampus, the mechanisms of gliotransmission need to be reconsidered.

Spontaneous neurotransmitter release and activation of group I metabotropic glutamate receptors (mGluRs) each play a role in the plasticity of neuronal synapses. Astrocytes may contribute to short-and long-term synaptic changes by signaling to neurons via these processes. Spontaneous whole-cell AMPA receptor (AMPAR) currents were recorded in CA1 pyramidal cells in situ while evoking Ca 2ϩ increases in the adjacent stratum radiatum astrocytes by uncaging IP 3 . Whole-cell patch clamp was used to deliver caged IP 3 and the Ca 2ϩ indicator dye Oregon green BAPTA-1 to astrocytes. Neurons were patch-clamped and filled with Alexa 568 hydrazide dye to visualize their morphological relationship to the astrocyte. On uncaging of IP 3 , astrocyte Ca 2ϩ responses reliably propagated as a wave into the very fine distal processes, synchronizing Ca 2ϩ activity within astrocyte microdomains. The intracellular astrocyte Ca 2ϩ wave coincided with a significant increase in the frequency of AMPA spontaneous EPSCs, but with no change in their kinetics. AMPAR current amplitudes were increased as well, but not significantly ( p ϭ 0.06). The increased frequency of AMPAR currents was sensitive to the group I mGluR antagonists LY367385 and 2-methyl-6-(phenylethynyl)-pyridine, suggesting that (1) astrocytes released glutamate in response to IP 3 uncaging, and (2) glutamate released by astrocytes activated group I mGluRs to facilitate the release of glutamate from excitatory neuronal presynaptic boutons. These results extend previous studies, which have shown astrocyte modulation of neuronal activity in vitro and suggest that astrocyte-to-neuron signaling in intact tissue may contribute to synaptic plasticity.

Astrocytes are considered the third component of the synapse, responding to neurotransmitter release from synaptic terminals and releasing gliotransmitters--including glutamate--in a Ca(2+)-dependent manner to affect neuronal synaptic activity. Many studies reporting astrocyte-driven neuronal activity have evoked astrocyte Ca(2+) increases by application of endogenous ligands that directly activate neuronal receptors, making astrocyte contribution to neuronal effect(s) difficult to determine. We have made transgenic mice that express a Gq-coupled receptor only in astrocytes to evoke astrocyte Ca(2+) increases using an agonist that does not bind endogenous receptors in brain. By recording from CA1 pyramidal cells in acute hippocampal slices from these mice, we demonstrate that widespread Ca(2+) elevations in 80%-90% of stratum radiatum astrocytes do not increase neuronal Ca(2+), produce neuronal slow inward currents, or affect excitatory synaptic activity. Our findings call into question the developing consensus that Ca(2+)-dependent glutamate release by astrocytes directly affects neuronal synaptic activity in situ.

2ϩ . Furthermore, neuronal G q -linked GPCR Ca 2ϩ increases remain intact, suggesting that IP 3 R2 does not play a major functional role in neuronal calcium store release or may not be expressed in neurons. Additionally, we show that lack of IP 3 R2 in the hippocampus does not affect baseline excitatory neuronal synaptic activity as measured by spontaneous EPSC recordings from CA1 pyramidal neurons. Whole-cell recordings of the tonic NMDA receptor-mediated current indicates that ambient glutamate levels are also unaffected in the IP 3 R2 KO. These data show that IP 3 R2 is the key functional IP 3 R driving G q -linked GPCR-mediated Ca 2ϩ increases in hippocampal astrocytes and that removal of astrocyte Ca 2ϩ increases does not significantly affect excitatory neuronal synaptic activity or ambient glutamate levels.

The possibility that astrocytes are involved in brain signaling began to emerge in the late 1970s, when it was first shown that astroglia in vitro possess numerous receptors for neurotransmitters. It was later demonstrated that cultured astroglia and astrocytes in situ respond to neurotransmitters with increases in intracellular second messengers, including cyclic AMP and calcium. Astrocyte calcium responses have since been extensively studied both in culture and in intact tissue. We continue to gather information regarding the various compounds able to trigger astrocyte calcium increases, as well as the mechanisms involved in their initiation, propagation as a calcium wave within and between astrocytes, and effects on signaling within the brain. This review will focus on each of these aspects of astrocyte calcium regulation, and attempt to sort out which effects are more likely to occur in developmental, pathological, and physiological conditions. While we have come far in our understanding of the properties or potential of astrocytes' ability to signal to neurons using our array of pharmacological tools, we still understand very little regarding the level of involvement of astrocyte signaling in normal brain physiology.

The immune privileged nature of the CNS can make it vulnerable to chronic and latent infections. Little is known about the effects of lifelong brain infections, and thus inflammation, on the neurological health of the host. Toxoplasma gondii is a parasite that can infect any mammalian nucleated cell with average worldwide seroprevalence rates of 30%. Infection by Toxoplasma is characterized by the lifelong presence of parasitic cysts within neurons in the brain, requiring a competent immune system to prevent parasite reactivation and encephalitis. In the immunocompetent individual, Toxoplasma infection is largely asymptomatic, however many recent studies suggest a strong correlation with certain neurodegenerative and psychiatric disorders. Here, we demonstrate a significant reduction in the primary astrocytic glutamate transporter, GLT-1, following infection with Toxoplasma. Using microdialysis of the murine frontal cortex over the course of infection, a significant increase in extracellular concentrations of glutamate is observed. Consistent with glutamate dysregulation, analysis of neurons reveal changes in morphology including a reduction in dendritic spines, VGlut1 and NeuN immunoreactivity. Furthermore, behavioral testing and EEG recordings point to significant changes in neuronal output. Finally, these changes in neuronal connectivity are dependent on infection-induced downregulation of GLT-1 as treatment with the ß-lactam antibiotic ceftriaxone, rescues extracellular glutamate concentrations, neuronal pathology and function. Altogether, these data demonstrate that following an infection with T. gondii, the delicate regulation of glutamate by astrocytes is disrupted and accounts for a range of deficits observed in chronic infection.

A major controversy persists within the field of glial biology concerning whether or not, under physiological conditions, neuronal activity leads to Ca-dependent release of neurotransmitters from astrocytes, a phenomenon known as gliotransmission. Our perspective is that, while we and others can apply techniques to cause gliotransmission, there is considerable evidence gathered using astrocyte-specific and more physiological approaches which suggests that gliotransmission is a pharmacological phenomenon rather than a physiological process. Approaches providing evidence against gliotransmission include stimulation of Gq-GPCRs expressed only in astrocytes, as well as removal of the primary proposed source of astrocyte Ca responsible for gliotransmission. These approaches contrast with those supportive of gliotransmission, which include mechanical stimulation, strong astrocytic depolarization using whole-cell patch-clamp or optogenetics, uncaging Ca or IP3, chelating Ca using BAPTA, and nonspecific bath application of agonists to receptors expressed by a multitude of cell types. These techniques are not subtle and therefore are not supportive of recent suggestions that gliotransmission requires very specific and delicate temporal and spatial requirements. Other evidence, including lack of propagating Ca waves between astrocytes in healthy tissue, lack of expression of vesicular release machinery, and the demise of the d-serine gliotransmission hypothesis, provides additional evidence against gliotransmission. Overall, the data suggest that Ca-dependent release of neurotransmitters is the province of neurons, not astrocytes, in the intact brain under physiological conditions..