Cendra Agulhon

What Is the Role of Astrocyte Calcium in Neurophysiology

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.

Selective Stimulation of Astrocyte Calcium In Situ Does Not Affect Neuronal Excitatory Synaptic Activity

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.

Sorting out astrocyte physiology from pharmacology.

A number of exciting findings have been made in astrocytes during the past 15 years that have led many researchers to redefine how the brain works. Astrocytes are now widely regarded as cells that propagate Ca(2+) over long distances in response to stimulation, and, similar to neurons, release transmitters (called gliotransmitters) in a Ca(2+)-dependent manner to modulate a host of important brain functions. Although these discoveries have been very exciting, it is essential to place them in the proper context of the approaches used to obtain them to determine their relevance to brain physiology. This review revisits the key observations made in astrocytes that greatly impact how they are thought to regulate brain function, including the existence of widespread propagating intercellular Ca(2+) waves, data suggesting that astrocytes signal to neurons through Ca(2+)-dependent release of glutamate, and evidence for the presence of vesicular machinery for the regulated exocytosis of gliotransmitters.

Calcium Signaling and Gliotransmission in Normal vs. Reactive Astrocytes

A prominent area of neuroscience research over the past 20 years has been the acute modulation of neuronal synaptic activity by Ca2+-dependent release of the transmitters ATP, D-serine, and glutamate (called gliotransmitters) by astrocytes. Although the physiological relevance of this mechanism is under debate, emerging evidence suggests that there are critical factors in addition to Ca2+ that are required for gliotransmitters to be released from astrocytes. Interestingly, these factors include activated microglia and the proinflammatory cytokine Tumor Necrosis Factor α (TNFα), chemotactic cytokine Stromal cell-Derived Factor-1α (SDF-1α), and inflammatory mediator prostaglandin E2 (PGE2). Of note, microglial activation and release of inflammatory molecules from activated microglia and reactive astrocytes can occur within minutes of a triggering stimulus. Therefore, activation of astrocytes by inflammatory molecules combined with Ca2+ elevations may lead to gliotransmitter release, and be an important step in the early sequence of events contributing to hyperexcitability, excitotoxicity, and neurodegeneration in the damaged or diseased brain. In this review, we will first examine evidence questioning Ca2+-dependent gliotransmitter release from astrocytes in healthy brain tissue, followed by a close examination of recent work suggesting that Ca2+-dependent gliotransmitter release occurs as an early event in the development of neurological disorders and neuroinflammatory and neurodegenerative diseases.

Visualization of local Ca2+ dynamics with genetically encoded bioluminescent reporters

Measurements of local Ca2+ signalling at different developmental stages and/or in specific cell types is important for understanding aspects of brain functioning. The use of light excitation in fluorescence imaging can cause phototoxicity, photobleaching and auto‐fluorescence. In contrast, bioluminescence does not require the input of radiative energy and can therefore be measured over long periods, with very high temporal resolution. Aequorin is a genetically encoded Ca2+‐sensitive bioluminescent protein, however, its low quantum yield prevents dynamic measurements of Ca2+ responses in single cells. To overcome this limitation, we recently reported the bi‐functional Ca2+ reporter gene, GFP‐aequorin (GA), which was developed specifically to improve the light output and stability of aequorin chimeras [V. Baubet, et al., (2000) PNAS, 97, 7260–7265]. In the current study, we have genetically targeted GA to different microdomains important in synaptic transmission, including to the mitochondrial matrix, endoplasmic reticulum, synaptic vesicles and to the postsynaptic density. We demonstrate that these reporters enable ‘real‐time’ measurements of subcellular Ca2+ changes in single mammalian neurons using bioluminescence. The high signal‐to‐noise ratio of these reporters is also important in that it affords the visualization of Ca2+ dynamics in cell–cell communication in neuronal cultures and tissue slices. Further, we demonstrate the utility of this approach in ex‐vivo preparations of mammalian retina, a paradigm in which external light input should be controlled. This represents a novel molecular imaging approach for non‐invasive monitoring of local Ca2+ dynamics and cellular communication in tissue or whole animal studies.

Modulation of the autonomic nervous system and behaviour by acute glial cell Gq protein-coupled receptor activation in vivo

•  The activation of glial Gq protein‐coupled receptor (Gq‐GPCR) signalling cascades broadly activates the autonomic nervous system •  The activation of glial Gq‐GPCR signalling cascades affects activity‐related behaviour.

New tools for investigating astrocyte-to-neuron communication

Gray matter protoplasmic astrocytes extend very thin processes and establish close contacts with synapses. It has been suggested that the release of neuroactive gliotransmitters at the tripartite synapse contributes to information processing. However, the concept of calcium (Ca2+)-dependent gliotransmitter release from astrocytes, and the release mechanisms are being debated. Studying astrocytes in their natural environment is challenging because: (i) astrocytes are electrically silent; (ii) astrocytes and neurons express an overlapping repertoire of transmembrane receptors; (iii) the size of astrocyte processes in contact with synapses are below the resolution of confocal and two-photon microscopes (iv) bulk-loading techniques using fluorescent Ca2+ indicators lack cellular specificity. In this review, we will discuss some limitations of conventional methodologies and highlight the interest of novel tools and approaches for studying gliotransmission. Genetically encoded Ca2+ indicators (GECIs), light-gated channels, and exogenous receptors are being developed to selectively read out and stimulate astrocyte activity. Our review discusses emerging perspectives on: (i) the complexity of astrocyte Ca2+ signaling revealed by GECIs; (ii) new pharmacogenetic and optogenetic approaches to activate specific Ca2+ signaling pathways in astrocytes; (iii) classical and new techniques to monitor vesicle fusion in cultured astrocytes; (iv) possible strategies to express specifically reporter genes in astrocytes.

Recent molecular approaches to understanding astrocyte function in vivo

Astrocytes are a predominant glial cell type in the nervous systems, and are becoming recognized as important mediators of normal brain function as well as neurodevelopmental, neurological, and neurodegenerative brain diseases. Although numerous potential mechanisms have been proposed to explain the role of astrocytes in the normal and diseased brain, research into the physiological relevance of these mechanisms in vivo is just beginning. In this review, we will summarize recent developments in innovative and powerful molecular approaches, including knockout mouse models, transgenic mouse models, and astrocyte-targeted gene transfer/expression, which have led to advances in understanding astrocyte biology in vivo that were heretofore inaccessible to experimentation. We will examine the recently improved understanding of the roles of astrocytes – with an emphasis on astrocyte signaling – in the context of both the healthy and diseased brain, discuss areas where the role of astrocytes remains debated, and suggest new research directions.

Bioluminescent imaging of Ca2+ activity reveals spatiotemporal dynamics in glial networks of dark-adapted mouse retina

Glial Ca2+ excitability plays a key role in reciprocal neuron–glia communication. In the retina, neuron–glia signalling is expected to be maximal in the dark, but the glial Ca2+ signal characteristics under such conditions have not been evaluated. To address this question, we used bioluminescence imaging to monitor spontaneous Ca2+ changes under dark conditions selectively in Müller cells, the principal retinal glial cells. By combining this imaging approach with network analysis, we demonstrate that activity in Müller cells is organized in networks of coactive cells, involving 2–16 cells located distantly and/or in clusters. We also report that spontaneous activity of small networks (2–6 Müller cells) repeat over time, sometimes in the same sequential order, revealing specific temporal dynamics. In addition, we show that networks of coactive glial cells are inhibited by TTX, indicating that ganglion and/or amacrine neuronal cells probably regulate Müller cell network properties. These results represent the first demonstration that spontaneous activity in adult Müller cells is patterned into correlated networks that display repeated sequences of coactivations over time. Furthermore, our bioluminescence technique provides a novel tool to study the dynamic characteristics of glial Ca2+ events in the retina under dark conditions, which should greatly facilitate future investigations of retinal dark‐adaptive processes.

AhR-deficiency as a cause of demyelinating disease and inflammation

The Aryl hydrocarbon Receptor(AhR) is among the most important receptors which bind pollutants; however it also regulates signaling pathways independently of such exposure. We previously demonstrated that AhR is expressed during development of the central nervous system(CNS) and that its deletion leads to the occurrence of a congenital nystagmus. Objectives of the present study are to decipher the origin of these deficits, and to identify the role of the AhR in the development of the CNS. We show that the AhR-knockout phenotype develops during early infancy together with deficits in visual-information-processing which are associated with an altered optic nerve myelin sheath, which exhibits modifications in its lipid composition and in the expression of myelin-associated-glycoprotein(MAG), a cell adhesion molecule involved in myelin-maintenance and glia-axon interaction. In addition, we show that the expression of pro-inflammatory cytokines is increased in the impaired optic nerve and confirm that inflammation is causally related with an AhR-dependent decreased expression of MAG. Overall, our findings demonstrate the role of the AhR as a physiological regulator of myelination and inflammatory processes in the developing CNS. It identifies a mechanism by which environmental pollutants might influence CNS myelination and suggest AhR as a relevant drug target for demyelinating diseases.