Synaptic information processing by astrocytes.

Glial cells were classically considered as supportive cells that do not contribute to information processing in the nervous system. However, considerable amount of evidence obtained by several groups during the last few years has demonstrated the existence of a bidirectional communication between astrocytes and neurons, which prompted a re-examination of the role of glial cells in the physiology of the nervous system. This review will discuss recent advances in the neuron-to-astrocyte communication, focusing on the recently reported properties of the synaptically evoked astrocyte Ca2+ signal that indicate that astrocytes show integrative properties for synaptic information processing. Indeed, we have recently shown that hippocampal astrocytes discriminate between the activity of different synapses, and respond selectively to different axon pathways. Furthermore, the astrocyte Ca2+ signal is modulated by the simultaneous activity of different synaptic inputs. This Ca2+ signal modulation depends on cellular intrinsic properties of the astrocytes, is bidirectionally regulated by the level of synaptic activity, and controls the spatial extension of the intracellular Ca2+ signal. Consequently, we propose that astrocytes can be considered as cellular elements involved in information processing by the nervous system.     

Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors

Fast excitatory neurotransmission is mediated by activation of synaptic ionotropic glutamate receptors. In hippocampal slices, we report that stimulation of Schaffer collaterals evokes in CA1 neurons delayed inward currents with slow kinetics, in addition to fast excitatory postsynaptic currents. Similar slow events also occur spontaneously, can still be observed when neuronal activity and synaptic glutamate release are blocked, and are found to be mediated by glutamate released from astrocytes acting preferentially on extrasynaptic NMDA receptors. The slow currents can be triggered by stimuli that evoke Ca2+ oscillations in astrocytes, including photolysis of caged Ca2+ in single astrocytes. As revealed by paired recording and Ca2+ imaging, a striking feature of this NMDA receptor response is that it occurs synchronously in multiple CA1 neurons. Our results reveal a distinct mechanism for neuronal excitation and synchrony and highlight a functional link between astrocytic glutamate and extrasynaptic NMDA receptors.     

The tripartite synapse: roles for gliotransmission in health and disease

In addition to being essential supporters of neuronal function, astrocytes are now recognized as active elements in the brain. Astrocytes sense and integrate synaptic activity and, depending on intracellular Ca(2+) levels, release gliotransmitters (e.g. glutamate, d-serine and ATP) that have feedback actions on neurons. Recent experimental results have raised the possibility that quantitative variations in gliotransmission might contribute to disorders of the nervous system. Here, we discuss targeted molecular genetic approaches that have demonstrated that alterations in protein expression in astrocytes can lead to serious changes in neuronal function. We also introduce the concept of 'astrocyte activation spectrum' in which enhanced and reduced gliotransmission might contribute to epilepsy and schizophrenia, respectively. The results of future experimental tests of the astrocyte activation spectrum, which relates gliotransmission to neurological and psychiatric disorders, might point to a new therapeutic target in the brain.     

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.     

Plasmin potentiates synaptic N-methyl-D-aspartate receptor function in hippocampal neurons through activation of protease-activated receptor-1

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC(50) 64 nm) and Fura-2/AM fluorescence (195 +/- 6.7% above base line, EC(50) 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the increase in phospho-ERK1/2 levels were diminished in PAR1(-/-) astrocytes and were blocked by 1 microm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca(2+)](i) signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-D-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or gamma-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 microm; 1.02 +/- 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca(2+)](i) in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.     

BDNF acutely modulates synaptic transmission and calcium signalling in developing cortical neurons.

Brain-derived neurotrophic factor (BDNF), like other neurotrophins, has long-term effects on neuronal survival and differentiation; furthermore, BDNF has been reported to exert an acute potentiation of synaptic activity and are critically involved in long-term potentiation(LTP). We found that BDNF rapidly induced potentiation of synaptic activity and an increase in the intracellular Ca2+ concentration in cultured cortical neurons. Within minutes of BDNF application to cultured cortical neurons, spontaneous firing rate was dramatically increased as was the frequency and amplitude of excitatory spontaneous postsynaptic currents (EPSCs). Fura-2 recordings showed that BDNF acutely elicited an increase in intracellular calcium concentration ([Ca2+]i). This effect was partially dependent on extracellular Ca2+. In calcium-free perfusion medium a substantial calcium signal remained which disappeared after loading of cortical neurons with 5 microM U-73122. BDNF-induce Ca2+ transients were completely blocked by K252a and partially blocked by Cd2+. The results demonstrate that BDNF can enhance synaptic transmission and induce directly a rise in [Ca2+]i that require two routes: the release of Ca2+ from intracellular calcium stores and influx of extracellular Ca2+ mainly through voltage-dependent Ca2+ channels in cultured cortical neurons.     

Reactive oxygen species mediate the potentiating effects of ATP on GABAergic synaptic transmission in the immature hippocampus

Reactive oxygen species (ROS) constitute important signaling molecules in the central nervous system. They regulate a number of different functions both under physiological conditions and under pathological conditions. Here we tested the hypothesis that in the immature hippocampus ATP, the most diffuse neurotransmitter in the brain, modulates synaptic transmission via ROS. We show that ATP, acting on metabotropic P2Y1 receptors, increased the frequency of GABA(A)-mediated spontaneous postsynaptic currents (SPSCs) in CA3 principal cells, an effect that was prevented by the antioxidant N-acetyl-cysteine or by catalase, an enzyme that breaks down H2O2. The effect of ATP on SPSCs was mimicked by H2O2 or by the pro-oxidant, Fe2+, which, through the Fentol reaction, catalyzes the conversion of H2O2 into highly reactive hydroxyl radicals. MRS-2179, a P2Y1 receptor antagonist, removed the facilitatory action of Fe2+ on SPSCs, suggesting that endogenous ATP acting on P2Y1 receptors is involved in Fe2+-induced modulation of synaptic transmission. Imaging ROS with the H2O2-sensitive dye DCF revealed that ATP induces generation of peroxide in astrocytes via activation of P2Y1 receptors coupled to intracellular calcium rise. Neither N-acetyl-cysteine nor catalase prevented Ca2+ transients induced by ATP in astrocytes. Since a single hippocampal astrocyte can contact many neurons, ATP-induced ROS signaling may control thousands of synapses. This may be crucial for information processing in the immature brain when GABAergic activity is essential for the proper wiring of the hippocampal network.     

Glutamate-mediated overexpression of CD38 in astrocytes cultured with neurones

Recently, a new system of astrocyte-neurone glutamatergic signalling has been identified. It is started in astrocytes by ectocellular, CD38-catalysed conversion of NAD(+) to the calcium mobilizer cyclic ADP-ribose (cADPR). This is then pumped by CD38 itself into the cytosol where the resulting free intracellular Ca(2+) concentration [Ca(2+)](i) transients elicit an increased release of glutamate, which can induce an enhanced Ca(2+) response in neighbouring neurones. Here, we demonstrate that co-culture of either cortical or hippocampal astrocytes with neurones results in a significant overexpression of astrocyte CD38 both on the plasma membrane and intracellularly. The causal role of neurone-released glutamate in inducing overexpression of astrocyte CD38 is demonstrated by two observations: first, in the absence of neurones, induction of CD38 in pure astrocyte cultures can be obtained with glutamate and second, it can be prevented in co-cultures by glutamate receptor antagonists. The neuronal glutamate-mediated effect of neurones on astrocyte CD38 expression is paralleled by increased intracellular cADPR and [Ca(2+)](i) levels, both findings indicating functionality of overexpressed CD38. These results reveal a new neurone-to-astrocyte glutamatergic signalling based on the CD38/cADPR system, which affects the [Ca(2+)](i) in both cell types, adding further complexity to the bi-directional patterns of communication between astrocytes and neurones.     

Calcium oscillations encoding neuron-to-astrocyte communication.

The observation that the excitatory neurotransmitter glutamate released from presynaptic terminals can activate, beside the post-synaptic neuron, the glial cell astrocyte, stimulated glial cell research like no other event since the recognition in the 1980s that astrocytes can express on their membrane many receptors for classical neurotransmitters. The properties and the functional role(s) of such a neuron-to-astrocyte signaling have now become the focus of intense research in neurobiology. Indeed, a growing body of evidence has recently highlighted the ability of astrocytes to work as sophisticated detectors of synaptic activity: by changing the frequency of [Ca(2+)](i) oscillations evoked by the synaptic release of glutamate, these cells display the remarkable capacity to discriminate between different levels and patterns of synaptic activity. Furthermore, the observation that astrocytes increase the frequency of [Ca(2+)](i) oscillations in response to repetitive episodes of high neuronal activity challenges the common concept that memory function in the brain is an exclusive property of neuronal cells. Glutamate-mediated [Ca(2+)](i) elevations can also trigger in astrocytes the release of glutamate that can ultimately affect neuronal transmission. Given the wide role played by glutamate in brain physiology, our view on how the brain operates needs now to be revised taking into account the bi-directional, glutamatergic communication between neurons and astrocytes.     

Exocytosis of ATP From Astrocytes Modulates Phasic and Tonic Inhibition in the Neocortex

Communication between neuronal and glial cells is important for many brain functions. Astrocytes can modulate synaptic strength via Ca²⁺-stimulated release of various gliotransmitters, including glutamate and ATP. A physiological role of ATP release from astrocytes was suggested by its contribution to glial Ca²⁺-waves and purinergic modulation of neuronal activity and sleep homeostasis. The mechanisms underlying release of gliotransmitters remain uncertain, and exocytosis is the most intriguing and debated pathway. We investigated release of ATP from acutely dissociated cortical astrocytes using “sniff-cell” approach and demonstrated that release is vesicular in nature and can be triggered by elevation of intracellular Ca²⁺ via metabotropic and ionotropic receptors or direct UV-uncaging. The exocytosis of ATP from neocortical astrocytes occurred in the millisecond time scale contrasting with much slower nonvesicular release of gliotransmitters via Best1 and TREK-1 channels, reported recently in hippocampus. Furthermore, we discovered that elevation of cytosolic Ca²⁺ in cortical astrocytes triggered the release of ATP that directly activated quantal purinergic currents in the pyramidal neurons. The glia-driven burst of purinergic currents in neurons was followed by significant attenuation of both synaptic and tonic inhibition. The Ca²⁺-entry through the neuronal P2X purinoreceptors led to phosphorylation-dependent down-regulation of GABAA receptors. The negative purinergic modulation of postsynaptic GABA receptors was accompanied by small presynaptic enhancement of GABA release. Glia-driven purinergic modulation of inhibitory transmission was not observed in neurons when astrocytes expressed dn-SNARE to impair exocytosis. The astrocyte-driven purinergic currents and glia-driven modulation of GABA receptors were significantly reduced in the P2X4 KO mice. Our data provide a key evidence to support the physiological importance of exocytosis of ATP from astrocytes in the neocortex.     

A tale of two stories: astrocyte regulation of synaptic depression and facilitation

Short-term presynaptic plasticity designates variations of the amplitude of synaptic information transfer whereby the amount of neurotransmitter released upon presynaptic stimulation changes over seconds as a function of the neuronal firing activity. While a consensus has emerged that the resulting decrease (depression) and/or increase (facilitation) of the synapse strength are crucial to neuronal computations, their modes of expression in vivo remain unclear. Recent experimental studies have reported that glial cells, particularly astrocytes in the hippocampus, are able to modulate short-term plasticity but the mechanism of such a modulation is poorly understood. Here, we investigate the characteristics of short-term plasticity modulation by astrocytes using a biophysically realistic computational model. Mean-field analysis of the model, supported by intensive numerical simulations, unravels that astrocytes may mediate counterintuitive effects. Depending on the expressed presynaptic signaling pathways, astrocytes may globally inhibit or potentiate the synapse: the amount of released neurotransmitter in the presence of the astrocyte is transiently smaller or larger than in its absence. But this global effect usually coexists with the opposite local effect on paired pulses: with release-decreasing astrocytes most paired pulses become facilitated, namely the amount of neurotransmitter released upon spike i+1 is larger than that at spike i, while paired-pulse depression becomes prominent under release-increasing astrocytes. Moreover, we show that the frequency of astrocytic intracellular Ca(2+) oscillations controls the effects of the astrocyte on short-term synaptic plasticity. Our model explains several experimental observations yet unsolved, and uncovers astrocytic gliotransmission as a possible transient switch between short-term paired-pulse depression and facilitation. This possibility has deep implications on the processing of neuronal spikes and resulting information transfer at synapses.     

The influence of the astrocyte field on neuronal dynamics and synchronization

Astrocytes can sense local synaptic release of glutamate by metabotropic glutamate receptors. Receptor activation in turn can mediate transient increases of astrocytic intracellular calcium concentration through inositol 1,4,5-trisphosphate production. Notably, the perturbation of calcium concentration can propagate to other adjacent astrocytes. Astrocytic calcium signaling can therefore be linked to synaptic information transfer between neurons. On the other hand, astrocytes can also modulate neuronal activity by feeding back onto synaptic terminals in a fashion that depends on their intracellular calcium concentration. Thus, astrocytes can also be active partners in neuronal network activity. The aim of our study is to provide a computationally simple network model of mutual neuron–astrocyte interactions, in order to investigate the possible roles of astrocytes in neuronal network dynamics. In particular, we focus on the information entropy of neuronal firing of the whole network, considering how it could be affected by neuron–glial interactions.     

Peptidomic analyses of mouse astrocytic cell lines and rat primary cultured astrocytes

Astrocytes play an active role in the modulation of synaptic transmission by releasing cell-cell signaling molecules in response to various stimuli that evoke a Ca(2+) increase. We expand on recent studies of astrocyte intracellular and secreted proteins by examining the astrocyte peptidome in mouse astrocytic cell lines and rat primary cultured astrocytes, as well as those peptides secreted from mouse astrocytic cell lines in response to Ca(2+)-dependent stimulations. We identified 57 peptides derived from 24 proteins with LC-MS/MS and CE-MS/MS in the astrocytes. Among the secreted peptides, four peptides derived from elongation factor 1, macrophage migration inhibitory factor, peroxiredoxin-5, and galectin-1 were putatively identified by mass-matching to peptides confirmed to be found in astrocytes. Other peptides in the secretion study were mass-matched to those found in prior peptidomics analyses on mouse brain tissue. Complex peptide profiles were observed after stimulation, suggesting that astrocytes are actively involved in peptide secretion. Twenty-six peptides were observed in multiple stimulation experiments but not in controls and thus appear to be released in a Ca(2+)-dependent manner. These results can be used in future investigations to better understand stimulus-dependent mechanisms of astrocyte peptide secretion.     

Neuronal activity triggers calcium waves in hippocampal astrocyte networks.

The recent discovery that the neurotransmitter glutamate can trigger actively propagating Ca2+ waves in the cytoplasm of cultured astrocytes suggests the possibility that synaptically released glutamate may trigger similar Ca2+ waves in brain astrocytes in situ. To explore this possibility, we used confocal microscopy and the Ca2+ indicator fluo-3 to study organotypically cultured slices of rat hippocampus, where astrocytic and neuronal networks are intermingled in their normal tissue relationships. We find that astrocytic Ca2+ waves are present under these circumstances and that these waves can be triggered by the firing of glutamatergic neuronal afferents with latencies as short as 2 s. The Ca2+ waves closely resemble those previously observed in cultured astrocytes: they propagate both within and between astrocytes at velocities of 7-27 microns/s at 21 degrees C. The ability of tissue astrocyte networks to respond to neuronal network activity suggests that astrocytes may have a much more dynamic and active role in brain function than has been generally recognized.     

Astrocytes and brain function: implications for reproduction

Recent evidence suggests that astrocytes have important neuroregulatory functions in addition to their classic functions of support and segregation of neurons. These newly revealed functions include regulation of neuron communication, neurosecretion, and synaptic plasticity. Although these actions occur throughout the brain, this review will focus on astrocyte-neuron interactions in the hypothalamus, particularly with respect to their potential contribution to the regulation of gonadotropin-releasing hormone (GnRH) secretion and reproduction. Hypothalamic astrocytes have been documented to release a variety of neuroactive factors, including transforming growth factors-alpha and -beta, insulin-like growth factor-1, prostaglandin E2, and the neurosteroid, 3 alpha-hydroxy-5 alpha-pregnane-20-one. Each of these factors has been shown to stimulate GnRH release, and receptors for each factor have been documented on GnRH neurons. Astrocytes have also been implicated in the regulation of synaptic plasticity in key areas of the hypothalamus that control GnRH release, an effect achieved by extension and retraction of glial processes (i.e., glial ensheathment). Through this mechanism, the number of synapses on GnRH neurons and GnRH regulatory neurons can potentially be modulated, thereby influencing the activation state of GnRH neurons. The steroid hormone 17beta-estradiol, which triggers the GnRH and luteinizing hormone surge, has been shown to induce the astrocyte-regulated changes in hypothalamic synaptic plasticity, as well as enhance formation and release of the astrocyte neuroactive factors, thereby providing another potential mechanistic layer for astrocyte regulation of GnRH release. As a whole, these studies provide new insights into the diversity of astrocytes and their potential role in reproductive neuroendocrine function.     

Synaptic plasticity and Ca2+ signalling in astrocytes

There is a growing body of evidence suggesting a functional relationship between Ca2+ signals generated in astroglia and the functioning of nearby excitatory synapses. Interference with endogenous Ca2+ homeostasis inside individual astrocytes has been shown to affect synaptic transmission and its use-dependent changes. However, establishing the causal link between source-specific, physiologically relevant intracellular Ca2+ signals, the astrocytic release machinery and the consequent effects on synaptic transmission has proved difficult. Improved methods of Ca2+ monitoring in situ will be essential for resolving the ambiguity in understanding the underlying Ca2+ signalling cascades.     

Activity-dependent neuronal control of gap-junctional communication in astrocytes

A typical feature of astrocytes is their high degree of intercellular communication through gap junction channels. Using different models of astrocyte cultures and astrocyte/neuron cocultures, we have demonstrated that neurons upregulate gap-junctional communication and the expression of connexin 43 (Cx43) in astrocytes. The propagation of intercellular calcium waves triggered in astrocytes by mechanical stimulation was also increased in cocultures. This facilitation depends on the age and number of neurons, indicating that the state of neuronal differentiation and neuron density constitute two crucial factors of this interaction. The effects of neurons on astrocytic communication and Cx43 expression were reversed completely after neurotoxic treatments. Moreover, the neuronal facilitation of glial coupling was suppressed, without change in Cx43 expression, after prolonged pharmacological treatments that prevented spontaneous synaptic activity. Altogether, these results demonstrate that neurons exert multiple and differential controls on astrocytic gap-junctional communication. Since astrocytes have been shown to facilitate synaptic efficacy, our findings suggest that neuronal and astrocytic networks interact actively through mutual setting of their respective modes of communication.     

Omega Conus geographus toxin: a peptide that blocks calcium channels

We previously reported that omega Conus geographus toxin (omega CgTX), blocks evoked-release of transmitter at synapses in frog and attenuates the Ca2+ component of the action potential of chick dorsal root ganglion neurons. We report here voltage-clamp experiments on cultured chick dorsal root ganglion neurons which demonstrate that omega CgTX produces a persistent block of voltage-gated Ca2+ currents. Thus, we conclude that omega CgTX inhibits synaptic transmission by blocking Ca2+ channels in the presynaptic nerve terminal. The toxin had no effect on K+ currents; however, in some but not all neurons, omega CgTX reduced Na+ currents by 10-25%. These findings suggest that omega CgTX should be useful as a probe to examine synaptic Ca2+ channels.     

Computational modeling of paroxysmal depolarization shifts in neurons induced by the glutamate release from astrocytes

Recent experimental studies have shown that astrocytes respond to external stimuli with a transient increase of the intracellular calcium concentration or can exhibit self-sustained spontaneous activity. Both evoked and spontaneous astrocytic calcium oscillations are accompanied by exocytosis of glutamate caged in astrocytes leading to paroxysmal depolarization shifts (PDS) in neighboring neurons. Here, we present a simple mathematical model of the interaction between astrocytes and neurons that is able to numerically reproduce the experimental results concerning the initiation of the PDS. The timing of glutamate release from the astrocyte is studied by means of a combined modeling of a vesicle cycle and the dynamics of SNARE-proteins. The neuronal slow inward currents (SICs), induced by the astrocytic glutamate and leading to PDS, are modeled via the activation of presynaptic glutamate receptors. The dependence of the bidirectional communication between neurons and astrocytes on the concentration of glutamate transporters is analyzed, as well. Our numerical results are in line with experimental findings showing that astrocyte can induce synchronous PDSs in neighboring neurons, resulting in a transient synchronous spiking activity.     

Glial perspectives of metabolic states during cerebral hypoxia--calcium regulation and metabolic energy

Cooperation between astrocytes and neurons is a unique interaction between two highly specialized cell types of the brain. Therefore, lack of nutrient supply during ischemia requires tight coordination of metabolism between astrocytes and neurons to keep the brain functions intact. To understand the impact of energy limitation on astrocytes, the functions of astrocytes have to be considered: (i) supplementation of neuronal cells, (ii) modulation of the extracellular milieu, mainly of the glutamate level, and (iii) elimination of reactive oxygen species (ROS). In cultured astrocytes and neurons inhibition of oxidative phosphorylation, using rotenone, was tested. Interestingly, this had only a negligible effect on Ca2+ homeostasis in astrocytes, even in combination with a severe glutamate stress. In contrast, in neurons glutamate in the presence of rotenone induced Ca2+ deregulation. Ca2+ homeostasis is very critical for cell survival. A massive and prolonged Ca2+ rise will lead to deregulation of many processes in such a way that the cells affected can hardly survive. Ca2+ homeostasis depends on the energy-consuming processes, which maintain the steep gradient between intracellular and extracellular Ca2+ concentration. Deprivation of oxygen and glucose during ischemia leads to a depletion of ATP in the brain, due to inhibited glycolytic and mitochondrial activity, whereas energy-consuming processes like ion pumps drain the ATP pools. On the other hand, specific mechanisms can protect brain structures against the massive insult of ischemia. Glycogen, stored in astrocytes, can maintain both neurons and astrocytes alive during short limitation of oxygen and glucose. Moreover, astrocytes can fuel ATP generation by providing lactate for neurons.