- Email: [email protected]
Astrocytic mGluR5 and the tripartite synapse
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Neuroscience xxx (2015) xxx–xxx
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REVIEW
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ASTROCYTIC mGLUR5s AND THE TRIPARTITE SYNAPSE
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A. PANATIER a,b* AND R. ROBITAILLE c,d
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Neurocentre Magendie, INSERM U862, Bordeaux, France
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Universite´ de Bordeaux, Bordeaux, France
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Conclusion Acknowledgments References
Abstract—In the brain, astrocytes occupy a key position between vessels and synapses. Among their numerous functions, these glial cells are key partners of neurons during synaptic transmission. Astrocytes detect transmitter release through receptors and transporters at the level of their processes, which are in close proximity to the tow neuronal elements of synapses. In response to transmittermediated activation, glial cells in turn regulate synaptic transmission and neuronal excitability. This process has been reported to involve several glial receptors. One of the best known of such receptors is the glutamatergic metabotropic receptor subtype 5 (mGluR5). In the present review we will discuss the implication of mGluR5s as detectors of synaptic transmission. In particular, we will discuss how the functional properties and localization of these receptors permit the detection of the synaptic signal in a defined temporal window and a given spatial area around the synapse. Furthermore, we will review the impact of their activation on synaptic transmission. This article is part of a Special Issue entitled: AstrocyteNeuron Interact. Ó 2015 Published by Elsevier Ltd. on behalf of IBRO.
Key words: astrocyte, mGluR5s, synaptic transmission, tripartite synapse. 13
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De´partement de neurosciences, Universite´ de Montre´al, PO Box 6128, Station centre-ville, Montre´al, Que´bec H3C 3J7, Canada
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Groupe de recherche sur le syste`me nerveux central, Universite´ de Montre´al, Canada
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Contents Introduction Properties of mGluR5s in astrocytes Expression of mGluR5s in astrocytes Astrocytes detect glutamatergic transmission through mGluR5s Astrocytes regulate basal synaptic transmission and neuronal synchrony through mglur5 activation
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*Correspondence to: A. Panatier, Neurocentre Magendie, INSERM U862, Bordeaux, France. E-mail address: [email protected] (A. Panatier). Abbreviations: GFAP, glial fibrillary acidic protein; IP3, 1,4,5-triphosphate; mGluR5, metabotropic receptor subtype 5; NMDA, N-methyl-d-aspartate; SICs, slow inward currents. http://dx.doi.org/10.1016/j.neuroscience.2015.03.063 0306-4522/Ó 2015 Published by Elsevier Ltd. on behalf of IBRO. 1
INTRODUCTION
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Increasing evidence indicates that astrocytes, the most abundant type of glial cells in the brain, participate actively in synaptic transmission. Indeed, owing to the ideal position of their processes around synapses (Ventura and Harris, 1999; Witcher et al., 2007), astrocytes are able to detect, and in turn regulate, the efficacy of synaptic transmission and neuronal excitability. Several mechanisms of regulation have been described, one of which is the release of active substances called gliotransmitters from astrocytes (Parpura et al., 2012; Araque et al., 2014; Rusakov et al., 2014). As a whole, accumulation of data since the 90s fostered the emergence of the concept of the tripartite synapse, in which astrocytes are considered as active partners of neurons at the synapse (Araque et al., 1999). Astrocytes detect synaptic activity through specialized receptors that are adapted to the type of synapses they interact with. One of the most prominent receptors implicated in the detection of neurotransmitters is the glutamatergic metabotropic receptor subtype 5 (mGluR5). Activation of this receptor leads to an increase of Ca2+ in astrocytes (Porter and McCarthy, 1995, 1996; Pasti et al., 1997; Latour et al., 2001; Wang et al., 2006; D’Ascenzo et al., 2007; Honsek et al., 2010; Panatier et al., 2011; Sun et al., 2014). Interestingly, the observed Ca2+ events occur with different spatial and temporal properties depending on the level of synaptic activity (Araque et al., 2014). This can range from a very local activity in a small portion of an astrocytic process that interacts with the two neuronal elements, called the astrocytic compartment (Panatier et al., 2011), up to the level of a response throughout the whole cell (Porter and McCarthy, 1995, 1996; Pasti et al., 1997; Latour et al., 2001; Wang et al., 2006; D’Ascenzo et al., 2007; Honsek et al., 2010; Sun et al., 2014). In this review we will discuss these different types of Ca2+ events as well as the distinct signaling integration and, ultimate regulation of synaptic transmission that may result from such events.
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PROPERTIES OF mGLUR5s IN ASTROCYTES
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mGluRs are a member of the large family of G-proteincoupled receptors. Their structure is composed of a large extracellular N-terminal domain, termed the Venus flytrap domain, containing the glutamate binding site. This domain is followed by seven transmembranespanning domains and an intracellular C terminus, a key region in the modulation of G protein coupling. The binding of glutamate leads to a modification in the conformation of the receptor and the activation of G proteins (Niswender and Conn, 2010). mGluRs are constitutive dimers organized into three groups, according to their G-protein coupling and signal transduction. Group I includes mGluRs 1 and 5, Group II includes mGluRs 2 and 3 and finally group III includes mGluRs 4, 6, 7, and 8. Group I mGluRs are primarily coupled to phospholipase C (PLC) while groups II and III are primarily coupled to adenyl cyclase (Tanabe et al., 1992; Niswender and Conn, 2010). In general, group I mGluRs are coupled to Gq/G11 G proteins and there activation leads to the activation of the phospholipase Cß, which hydrolyzes membrane phosphoinositides to form inositol 1,4,5-triphosphate (IP3) (Niswender and Conn, 2010), activates of Protein Kinase C (PKC) and ultimately mobilizes Ca2+ from endoplasmic reticulum and activates a plethora of Ca2+dependent mechanisms (Niswender and Conn, 2010). A precise localization and interaction must exist between mGluRs and the molecular elements of their activated cascades. For instance, in neurons it has been shown that Shank1B and Homer1b, two scaffold proteins, are required for mGluR5-mediated IP3 generation and thus Ca2+ release from internal stores (Sala et al., 2005). Furthermore, homer is important for the localization of mGluR5s as well as for the assembly of protein complexes at the C-terminal tails of mGluR5s in neurons (Niswender and Conn, 2010). Interestingly, a study performed on hypothalamic and cerebrocortical astrocytic cultures revealed the presence of a similar physical association between mGluR5s and Homer in astrocytes (Dziedzic et al., 2003). However, whether Homer allows mGluR5s to be compartmentalized with the endoplasmic reticulum in astrocytes remains unknown. Additionally, a study performed on pure astrocytic cultures from rat neocortices revealed that astrocytic mGluR5s show a similar affinity for agonists to that of mGluR5s expressed in cell lines and Xenopus oocytes (Balazs et al., 1997). This study also found that activation of astrocytic mGluR5s, only leads to the hydrolysis of phosphoinositides (Balazs et al., 1997). An important property of astrocytic mGluR5s is their reduced desensitization in comparison to neuronal mGluR5s. Indeed, in astrocytes, mGluR5 activity was unaltered by a 15-min exposure to the mGluR agonist ACPD. Furthermore, mGluR5 activity was decreased by only 20–30% after a long incubation (4 h) of the agonist (Balazs et al., 1997). In addition, although mGluR5 density has been reported to be lower in astrocytes when compared to that of neurons (about one-fifth) (Brabet et al., 1995; Balazs et al., 1997), astrocyte receptors have
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a much higher efficiency to induce phosphoinositides hydrolysis (Balazs et al., 1997). Hence, these properties make them ideally suited to detect with high efficacy and reliability small increases in synaptic glutamate. Finally, it is of importance to note that the level of mGluR5s mRNA decreases in adulthood (Cai et al., 2000; Morel et al., 2014) while mGluR3 level remains stable (Schools and Kimelberg, 1999; Sun et al., 2013).
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EXPRESSION OF mGLUR5s IN ASTROCYTES
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Experiments performed on acutely isolated astrocytes revealed that the mRNA expression of mGluR5s varies throughout development (Schools and Kimelberg, 1999; Cai et al., 2000; Sun et al., 2013; Morel et al., 2014). In the hippocampus, from post-natal days (P) 1–10, 58% of glial fibrillary acidic protein (GFAP)-positive cells expressed mGluR5 mRNAs while 78% of the cells expressed mGluR3 mRNAs (Schools and Kimelberg, 1999). In another study, the same group also reported that 77% of GFAP-positive cells expressed mGluR5 mRNA in mice at age P11–P20 (Cai et al., 2000). Finally, in more mature mice, from P25 to P35, mGluR5 mRNAs were only found in 36% of astrocytes (Cai et al., 2000).These values may be an underestimate since, in this study, acute cells from P25 to P35 hippocampi rarely showed processes in comparison to cells isolated from younger animals (Cai et al., 2000). This is truly of note as the number and complexity of astrocyte processes is known to increase with age (Bushong et al., 2004). Hence, it appears that a large proportion of astrocyte processes from adult animals were not extracted. This is quite important since mGluR5s are likely to be located in these fine processes, close to synapses (Panatier et al., 2011). A more accurate estimate may therefore be obtained by performing single cell RT-PCR directly on acute slices. Furthermore, one must also consider the physiological activity of these receptors rather than solely their level of mRNA expression. For instance, a similar situation is found when considering the level of expression of CB1cannabinoid receptors, which are expressed at very high levels in certain brain cell types and at very low levels in others, such as astrocytes. However, these low levels of astroglial CB1 receptors provide profound and important regulations at multiple levels (Han et al., 2012; Metna-Laurent and Marsicano, 2014).
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ASTROCYTES DETECT GLUTAMATERGIC TRANSMISSION THROUGH mGLUR5s
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As a first step in the discovery and understanding of astrocyte excitability, it has been observed that astrocytes in culture detect glutamate applied in the petri dish (Bowman and Kimelberg, 1984; Glaum et al., 1990; Cornell-Bell et al., 1990a,b). Interestingly a low concentration of glutamate (<1 lM) induces Ca2+ events asynchronously in distinct parts of a single astrocyte (Cornell-Bell et al., 1990a) while higher concentrations of glutamate induce an initial increase of Ca2+ in multiple cells followed by oscillations (Cornell-Bell et al., 1990a). These evoked Ca2+ events are in part due to
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Ca2+ release from intracellular stores (Cornell-Bell et al., 1990a). Importantly, the kinetics of these Ca2+ events were dependent on the concentration and duration of glutamate application, in a non-predictive direction. Indeed, average periods of oscillations decreased as the concentration and application time of glutamate were increased (Cornell-Bell et al., 1990a). Is this decreased response due to an integrating process? Using acute hippocampal slices, Perea and Araque have shown that astrocytes can discriminate between the activity of different synapses, suggesting an integrating process in the astrocyte (Perea and Araque, 2005). To advance on this work, it would be interesting to investigate whether the activity in one region of the astrocyte, influences the activity in another region of the same cell. In a more integrated system, application of the mGluRs agonist induces a Ca2+ increase in CA1 astrocytes of acute hippocampal slices prepared from young animals (P9–P13) (Porter and McCarthy, 1995). Importantly, it was later shown that astrocytes are able to detect glutamate release during synaptic transmission induced by a train of stimuli (Porter and McCarthy, 1996; Latour et al., 2001). This detection of synaptic activity by astrocytes was dependent on the activation of group I/II mGluRs, most likely mGluR5s since mGluR1s do not appear to be expressed by hippocampal astrocytes (Schools and Kimelberg, 1999). The detection of synaptic activity by astrocytes is not all or none but rather is dependent on the pattern of activity of synaptic transmission (Pasti et al., 1997) such that the amplitude of Ca2+ oscillations are dependent on the number of synapses recruited. Furthermore, these oscillations are potentiated by activation of mGluRs with t-ACPD (Pasti et al., 1997). Finally, using a specific antagonist for mGluR5s (MPEP), it has been shown recently that mGluR5s are the receptors implicated in the increase in astrocytic Ca2+ observed during synaptic transmission (Honsek et al., 2010; Panatier et al., 2011; Sun et al., 2014). Until recently, reports indicated that astrocytic Ca2+ events were only induced by a train of synaptic activity. If that were the case astrocytes would remain completely oblivious to a large array of synaptic information that is conveyed at lower and less intense levels of activity. To address whether astrocytes play a crucial role during basal synaptic transmission, we tested whether astrocytes could detect glutamate released during synaptic transmission at the level of an individual synapse, induced by a single pulse (Panatier et al., 2011). Our work revealed that, in juvenile rats, astrocytes detect basal synaptic transmission at the single synapse level, simultaneously with the post-synaptic element, through mGluR5s detection (Panatier et al., 2011). Small and rapid Ca2+ elevations were observed in small astrocytic compartments adjacent to the dendritic spines. We concluded that these data further extend the capacity of astrocytes in the detection and integration of synaptic transmission. The fact that astrocytes detect the lowest level of synaptic communication raises several questions. For instance, what happens when individual Ca2+ events are induced at the level of neighboring synapses? Is there a threshold of activity necessary to
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transform the local glial response to a larger and spatially extended response? Is this level of sensitivity also occurring for other types of synapses such as GABAergic or cholinergic synapses? Interestingly, another group was unable to record these small and localized responses at the same synapses (hippocampal CA3–CA1 synapses) (Sun et al., 2014). However, there are two fundamental spatio-temporal issues that were not optimal in this work. First, it appears that the interval between scans (0.3-s scanning interval, 3.3 Hz) was too slow. Indeed, the short duration of the local Ca2+ responses requires the use of a rate of acquisition much faster than the one used by these authors. An insufficient acquisition rate may lead to the failure to record fast and small events. Second, the Ca2+ responses occur in small spatially constrained compartments (size around 1-lm length maximum) that are associated with dendritic spines and synapses. Owing to the complex, spongiform morphology of an astrocyte, it is difficult to perform imaging at the proper/ideal location. The temporally and spatially intimate relationship between synapses and their activity must be taken into consideration as they dictate the location and timing of the glial response to the minimal release of glutamate. As a whole, it is crucial that the acquisition be performed at a confirmed synaptic site using rapid acquisition rates (e.g. using the line scan mode of a confocal) (Panatier et al., 2011). Additional technical details can be found in (Panatier and Robitaille, 2013). In addition to their roles in the hippocampus, astrocytic mGluR5s also play crucial roles in the detection of synaptic transmission in the thalamus of young rats (Parri et al., 2010), in the nucleus accumbens (D’Ascenzo et al., 2007) and cortex of adult mice (Wang et al., 2006). Nedergaard and coworkers observed robust astrocytic Ca2+ events in response to in vivo tACPD application by iontophoresis in layer 2 of the barrel cortex in adult mice (Wang et al., 2006). More importantly, MPEP-sensitive astrocytic Ca2+ events were evoked by whisker stimulation in a frequency-dependent manner (Wang et al., 2006). However, in a more recent study, the same group failed to induce Ca2+ elevation in astrocytes of adult mice in vivo. These studies used a less well-controlled application of agonists co-injected with Alexa 488. More importantly, the magnification used to perform Ca2+ imaging was lower (Sun et al., 2013), implying that activity within the astrocytic processes where mGluR5 are preferentially located could have easily been missed. As a whole, data from Wang and collaborators are more supportive of the presence and expression of mGluR5s by cortical astrocytes in adults. However, their implication in the activation of astrocytes is in need of a systematic and careful analysis.
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Astrocytes are characterized by the presence of stubby main processes and dense ramifications of thinner processes with an irregular shape, giving them a
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spongiform morphology (Bushong et al., 2002, 2004). In the hippocampus (Bushong et al., 2002) and the cortex (Halassa et al., 2007) a single astrocyte has its own, non-overlapping territory, named the astrocytic domain. It has been estimated that each astrocyte could contact around 100,000 synapses (Bushong et al., 2002), and 4–8 neurons, encompassing 300–600 dendrites (Halassa et al., 2007). Hence, similarly to neurons, there are a large number of synapses that a given astrocyte is associated with. Accordingly, from a morphological to a functional point of view, astrocytes are well positioned to regulate synaptic transmission from a single synapse level (Panatier et al., 2011) to a neuronal network level (Araque et al., 2014). There are many neuronal and synaptic mechanisms that are regulated by astrocytes as a consequence of their synaptic activation. A number of these mechanisms are regulated by the activation of the glial mGluR5s. For instance, astrocytes detect synaptic transmission occurring at the single synapse level induced by minimal synaptic stimulation in the juvenile CA1 hippocampus (Panatier et al., 2011). Importantly, because of the electrophysiological tools and protocols used, only a single fiber was stimulated, leading to the activation of 1 to 2–3 synapses (Panatier et al., 2011).
Following this detection through mGluR5s, astrocytes release purines, which act on presynaptic adenosine A2A receptors to up-regulate basal synaptic transmission (Fig. 1, left). It is noteworthy that CA3–CA1 hippocampal synapses are tonically inhibited by astrocytic purines through the activation of another type of adenosine receptor, the presynaptic A1 receptor (Pascual et al., 2005). This interaction between A1R mediated tonic suppression of synaptic transmission and A2A-dependent up-regulation of synaptic transmission provides a mechanism through which synapses may be depressed or potentiated as a function of the activity in the network. In addition to purines there is also evidence that astrocytes release glutamate following stronger mGluR5 activation probably in a non-vesicular mode, as suggested by the absence of effect of tetanus toxin (Fig. 1, right) (Pasti et al., 1997; Angulo et al., 2004; Fellin et al., 2004). These authors reported that application of mGluR agonists induced the release of glutamate from astrocytes that in turn activates neighboring neuronal extrasynaptic glutamatergic N-methyl-D-aspartate (NMDA) receptors (Fig. 1, right) which induces an increase of neuronal Ca2+ (Pasti et al., 1997; Angulo et al., 2004; Fellin et al., 2004). Interestingly, the astrocytic glutamate activates specifically extrasynaptic
Fig. 1. Model of mGluR5-dependant neuronal-astrocyte interaction at hippocampal CA3–CA1 synapses. (Left) At the single synapse level, glutamate released after the arrival of a single action potential (1) activates astrocytic mGluR5s (2), leading to a local increase of Ca2+ in the astrocytic process (3). Subsequently Ca2+ triggers purine release. Once released, purines activate presynaptic A2A receptors (4), inducing an upregulation of the efficacy of transmission (5). (Right) During a sustained synaptic activity (1), glutamate activates astrocytic mGluR5s (2) leading to a large increase of Ca2+ (3) that propagates over a large portion of the astrocyte. Under this condition, glutamate is released from the astrocyte, activating extrasynaptic NMDARs (4). This release of glutamate from astrocyte controls neuronal excitability and synchrony (5). Please cite this article in press as: Panatier A, Robitaille R. Astrocytic mglur5s and the tripartite synapse. neuroscience (2015), http://dx.doi.org/ 10.1016/j.neuroscience.2015.03.063
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NMDA receptors (with NR2B subunit) in 2–4 neighboring neurons (Fellin et al., 2004). These NR2B-mediatied currents are called slow inward currents (SICs) (Fellin et al., 2004), or slow transient currents (STCs) (Angulo et al., 2004). They can either occur spontaneously at a very low frequency (Angulo et al., 2004; Fellin et al., 2004), or be evoked by Schaffer collateral stimulations (Fellin et al., 2004). Interestingly, these SICs could be synchronized within a time window of 100 ms in recorded neuronal pairs. Using this process, astrocytes are able to regulate neuronal excitability and synchrony (Angulo et al., 2004; Fellin et al., 2004). Importantly, a similar process has been suggested in the nucleus accumbens (D’Ascenzo et al., 2007).
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CONCLUSION
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The localization and density of mGluR5s will influence the integration of synaptic transmission by astrocytes and, thus, will be dependent on the level of synaptic activation. As with other receptors in the brain, mGluR5 expression is regulated during development. To better understand the role of mGluR5s it is important to not consider only its expression but its localization, properties and functions must also be taken into account. Furthermore, the space and time between synapses and their activity are fundamental. Thus, it is crucial to adapt the time and space domains of the experimental paradigm to fit the kinetics and properties of the astrocytes in a given physiological condition. Astrocytic mGluR5s are playing fundamental roles in the detection of synaptic signals and thus, in turn, in the regulation of synaptic transmission. This has been shown particularly at hippocampal CA3–CA1 synapses. Major advances in the roles of mGluR5s will rely upon a better understanding of the molecular machinery in particular the mGluR5s scaffolding at synapses and their organization along processes in association with the internal stores of Ca2+.
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Acknowledgments—We would like to thank Dr Clare Reynell for her comments on the manuscript. This work was supported by grants from Marie Curie FP7 (FP7-PEOPLE-2010-RG) and CNRS to AP. RR is a recipient of grants from the Canadian Institutes for Health research to (MOP-14137 and MOP111070), a Leader Opportunity Fund from the Canadian Foundation of Innovation and an infrastructure grant from Fonds de Recherche du Que´bec - Sante´ (FRQ - Sante´) to the GRSNC (Groupe de recherche sur le syste`me nerveux central).
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(Accepted 26 March 2015) (Available online xxxx)
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REVIEW
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ASTROCYTIC mGLUR5s AND THE TRIPARTITE SYNAPSE
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A. PANATIER a,b* AND R. ROBITAILLE c,d
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Neurocentre Magendie, INSERM U862, Bordeaux, France
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Universite´ de Bordeaux, Bordeaux, France
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Conclusion Acknowledgments References
Abstract—In the brain, astrocytes occupy a key position between vessels and synapses. Among their numerous functions, these glial cells are key partners of neurons during synaptic transmission. Astrocytes detect transmitter release through receptors and transporters at the level of their processes, which are in close proximity to the tow neuronal elements of synapses. In response to transmittermediated activation, glial cells in turn regulate synaptic transmission and neuronal excitability. This process has been reported to involve several glial receptors. One of the best known of such receptors is the glutamatergic metabotropic receptor subtype 5 (mGluR5). In the present review we will discuss the implication of mGluR5s as detectors of synaptic transmission. In particular, we will discuss how the functional properties and localization of these receptors permit the detection of the synaptic signal in a defined temporal window and a given spatial area around the synapse. Furthermore, we will review the impact of their activation on synaptic transmission. This article is part of a Special Issue entitled: AstrocyteNeuron Interact. Ó 2015 Published by Elsevier Ltd. on behalf of IBRO.
Key words: astrocyte, mGluR5s, synaptic transmission, tripartite synapse. 13
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De´partement de neurosciences, Universite´ de Montre´al, PO Box 6128, Station centre-ville, Montre´al, Que´bec H3C 3J7, Canada
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Groupe de recherche sur le syste`me nerveux central, Universite´ de Montre´al, Canada
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Contents Introduction Properties of mGluR5s in astrocytes Expression of mGluR5s in astrocytes Astrocytes detect glutamatergic transmission through mGluR5s Astrocytes regulate basal synaptic transmission and neuronal synchrony through mglur5 activation
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*Correspondence to: A. Panatier, Neurocentre Magendie, INSERM U862, Bordeaux, France. E-mail address: [email protected] (A. Panatier). Abbreviations: GFAP, glial fibrillary acidic protein; IP3, 1,4,5-triphosphate; mGluR5, metabotropic receptor subtype 5; NMDA, N-methyl-d-aspartate; SICs, slow inward currents. http://dx.doi.org/10.1016/j.neuroscience.2015.03.063 0306-4522/Ó 2015 Published by Elsevier Ltd. on behalf of IBRO. 1
INTRODUCTION
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Increasing evidence indicates that astrocytes, the most abundant type of glial cells in the brain, participate actively in synaptic transmission. Indeed, owing to the ideal position of their processes around synapses (Ventura and Harris, 1999; Witcher et al., 2007), astrocytes are able to detect, and in turn regulate, the efficacy of synaptic transmission and neuronal excitability. Several mechanisms of regulation have been described, one of which is the release of active substances called gliotransmitters from astrocytes (Parpura et al., 2012; Araque et al., 2014; Rusakov et al., 2014). As a whole, accumulation of data since the 90s fostered the emergence of the concept of the tripartite synapse, in which astrocytes are considered as active partners of neurons at the synapse (Araque et al., 1999). Astrocytes detect synaptic activity through specialized receptors that are adapted to the type of synapses they interact with. One of the most prominent receptors implicated in the detection of neurotransmitters is the glutamatergic metabotropic receptor subtype 5 (mGluR5). Activation of this receptor leads to an increase of Ca2+ in astrocytes (Porter and McCarthy, 1995, 1996; Pasti et al., 1997; Latour et al., 2001; Wang et al., 2006; D’Ascenzo et al., 2007; Honsek et al., 2010; Panatier et al., 2011; Sun et al., 2014). Interestingly, the observed Ca2+ events occur with different spatial and temporal properties depending on the level of synaptic activity (Araque et al., 2014). This can range from a very local activity in a small portion of an astrocytic process that interacts with the two neuronal elements, called the astrocytic compartment (Panatier et al., 2011), up to the level of a response throughout the whole cell (Porter and McCarthy, 1995, 1996; Pasti et al., 1997; Latour et al., 2001; Wang et al., 2006; D’Ascenzo et al., 2007; Honsek et al., 2010; Sun et al., 2014). In this review we will discuss these different types of Ca2+ events as well as the distinct signaling integration and, ultimate regulation of synaptic transmission that may result from such events.
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PROPERTIES OF mGLUR5s IN ASTROCYTES
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mGluRs are a member of the large family of G-proteincoupled receptors. Their structure is composed of a large extracellular N-terminal domain, termed the Venus flytrap domain, containing the glutamate binding site. This domain is followed by seven transmembranespanning domains and an intracellular C terminus, a key region in the modulation of G protein coupling. The binding of glutamate leads to a modification in the conformation of the receptor and the activation of G proteins (Niswender and Conn, 2010). mGluRs are constitutive dimers organized into three groups, according to their G-protein coupling and signal transduction. Group I includes mGluRs 1 and 5, Group II includes mGluRs 2 and 3 and finally group III includes mGluRs 4, 6, 7, and 8. Group I mGluRs are primarily coupled to phospholipase C (PLC) while groups II and III are primarily coupled to adenyl cyclase (Tanabe et al., 1992; Niswender and Conn, 2010). In general, group I mGluRs are coupled to Gq/G11 G proteins and there activation leads to the activation of the phospholipase Cß, which hydrolyzes membrane phosphoinositides to form inositol 1,4,5-triphosphate (IP3) (Niswender and Conn, 2010), activates of Protein Kinase C (PKC) and ultimately mobilizes Ca2+ from endoplasmic reticulum and activates a plethora of Ca2+dependent mechanisms (Niswender and Conn, 2010). A precise localization and interaction must exist between mGluRs and the molecular elements of their activated cascades. For instance, in neurons it has been shown that Shank1B and Homer1b, two scaffold proteins, are required for mGluR5-mediated IP3 generation and thus Ca2+ release from internal stores (Sala et al., 2005). Furthermore, homer is important for the localization of mGluR5s as well as for the assembly of protein complexes at the C-terminal tails of mGluR5s in neurons (Niswender and Conn, 2010). Interestingly, a study performed on hypothalamic and cerebrocortical astrocytic cultures revealed the presence of a similar physical association between mGluR5s and Homer in astrocytes (Dziedzic et al., 2003). However, whether Homer allows mGluR5s to be compartmentalized with the endoplasmic reticulum in astrocytes remains unknown. Additionally, a study performed on pure astrocytic cultures from rat neocortices revealed that astrocytic mGluR5s show a similar affinity for agonists to that of mGluR5s expressed in cell lines and Xenopus oocytes (Balazs et al., 1997). This study also found that activation of astrocytic mGluR5s, only leads to the hydrolysis of phosphoinositides (Balazs et al., 1997). An important property of astrocytic mGluR5s is their reduced desensitization in comparison to neuronal mGluR5s. Indeed, in astrocytes, mGluR5 activity was unaltered by a 15-min exposure to the mGluR agonist ACPD. Furthermore, mGluR5 activity was decreased by only 20–30% after a long incubation (4 h) of the agonist (Balazs et al., 1997). In addition, although mGluR5 density has been reported to be lower in astrocytes when compared to that of neurons (about one-fifth) (Brabet et al., 1995; Balazs et al., 1997), astrocyte receptors have
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a much higher efficiency to induce phosphoinositides hydrolysis (Balazs et al., 1997). Hence, these properties make them ideally suited to detect with high efficacy and reliability small increases in synaptic glutamate. Finally, it is of importance to note that the level of mGluR5s mRNA decreases in adulthood (Cai et al., 2000; Morel et al., 2014) while mGluR3 level remains stable (Schools and Kimelberg, 1999; Sun et al., 2013).
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EXPRESSION OF mGLUR5s IN ASTROCYTES
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Experiments performed on acutely isolated astrocytes revealed that the mRNA expression of mGluR5s varies throughout development (Schools and Kimelberg, 1999; Cai et al., 2000; Sun et al., 2013; Morel et al., 2014). In the hippocampus, from post-natal days (P) 1–10, 58% of glial fibrillary acidic protein (GFAP)-positive cells expressed mGluR5 mRNAs while 78% of the cells expressed mGluR3 mRNAs (Schools and Kimelberg, 1999). In another study, the same group also reported that 77% of GFAP-positive cells expressed mGluR5 mRNA in mice at age P11–P20 (Cai et al., 2000). Finally, in more mature mice, from P25 to P35, mGluR5 mRNAs were only found in 36% of astrocytes (Cai et al., 2000).These values may be an underestimate since, in this study, acute cells from P25 to P35 hippocampi rarely showed processes in comparison to cells isolated from younger animals (Cai et al., 2000). This is truly of note as the number and complexity of astrocyte processes is known to increase with age (Bushong et al., 2004). Hence, it appears that a large proportion of astrocyte processes from adult animals were not extracted. This is quite important since mGluR5s are likely to be located in these fine processes, close to synapses (Panatier et al., 2011). A more accurate estimate may therefore be obtained by performing single cell RT-PCR directly on acute slices. Furthermore, one must also consider the physiological activity of these receptors rather than solely their level of mRNA expression. For instance, a similar situation is found when considering the level of expression of CB1cannabinoid receptors, which are expressed at very high levels in certain brain cell types and at very low levels in others, such as astrocytes. However, these low levels of astroglial CB1 receptors provide profound and important regulations at multiple levels (Han et al., 2012; Metna-Laurent and Marsicano, 2014).
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ASTROCYTES DETECT GLUTAMATERGIC TRANSMISSION THROUGH mGLUR5s
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As a first step in the discovery and understanding of astrocyte excitability, it has been observed that astrocytes in culture detect glutamate applied in the petri dish (Bowman and Kimelberg, 1984; Glaum et al., 1990; Cornell-Bell et al., 1990a,b). Interestingly a low concentration of glutamate (<1 lM) induces Ca2+ events asynchronously in distinct parts of a single astrocyte (Cornell-Bell et al., 1990a) while higher concentrations of glutamate induce an initial increase of Ca2+ in multiple cells followed by oscillations (Cornell-Bell et al., 1990a). These evoked Ca2+ events are in part due to
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Ca2+ release from intracellular stores (Cornell-Bell et al., 1990a). Importantly, the kinetics of these Ca2+ events were dependent on the concentration and duration of glutamate application, in a non-predictive direction. Indeed, average periods of oscillations decreased as the concentration and application time of glutamate were increased (Cornell-Bell et al., 1990a). Is this decreased response due to an integrating process? Using acute hippocampal slices, Perea and Araque have shown that astrocytes can discriminate between the activity of different synapses, suggesting an integrating process in the astrocyte (Perea and Araque, 2005). To advance on this work, it would be interesting to investigate whether the activity in one region of the astrocyte, influences the activity in another region of the same cell. In a more integrated system, application of the mGluRs agonist induces a Ca2+ increase in CA1 astrocytes of acute hippocampal slices prepared from young animals (P9–P13) (Porter and McCarthy, 1995). Importantly, it was later shown that astrocytes are able to detect glutamate release during synaptic transmission induced by a train of stimuli (Porter and McCarthy, 1996; Latour et al., 2001). This detection of synaptic activity by astrocytes was dependent on the activation of group I/II mGluRs, most likely mGluR5s since mGluR1s do not appear to be expressed by hippocampal astrocytes (Schools and Kimelberg, 1999). The detection of synaptic activity by astrocytes is not all or none but rather is dependent on the pattern of activity of synaptic transmission (Pasti et al., 1997) such that the amplitude of Ca2+ oscillations are dependent on the number of synapses recruited. Furthermore, these oscillations are potentiated by activation of mGluRs with t-ACPD (Pasti et al., 1997). Finally, using a specific antagonist for mGluR5s (MPEP), it has been shown recently that mGluR5s are the receptors implicated in the increase in astrocytic Ca2+ observed during synaptic transmission (Honsek et al., 2010; Panatier et al., 2011; Sun et al., 2014). Until recently, reports indicated that astrocytic Ca2+ events were only induced by a train of synaptic activity. If that were the case astrocytes would remain completely oblivious to a large array of synaptic information that is conveyed at lower and less intense levels of activity. To address whether astrocytes play a crucial role during basal synaptic transmission, we tested whether astrocytes could detect glutamate released during synaptic transmission at the level of an individual synapse, induced by a single pulse (Panatier et al., 2011). Our work revealed that, in juvenile rats, astrocytes detect basal synaptic transmission at the single synapse level, simultaneously with the post-synaptic element, through mGluR5s detection (Panatier et al., 2011). Small and rapid Ca2+ elevations were observed in small astrocytic compartments adjacent to the dendritic spines. We concluded that these data further extend the capacity of astrocytes in the detection and integration of synaptic transmission. The fact that astrocytes detect the lowest level of synaptic communication raises several questions. For instance, what happens when individual Ca2+ events are induced at the level of neighboring synapses? Is there a threshold of activity necessary to
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transform the local glial response to a larger and spatially extended response? Is this level of sensitivity also occurring for other types of synapses such as GABAergic or cholinergic synapses? Interestingly, another group was unable to record these small and localized responses at the same synapses (hippocampal CA3–CA1 synapses) (Sun et al., 2014). However, there are two fundamental spatio-temporal issues that were not optimal in this work. First, it appears that the interval between scans (0.3-s scanning interval, 3.3 Hz) was too slow. Indeed, the short duration of the local Ca2+ responses requires the use of a rate of acquisition much faster than the one used by these authors. An insufficient acquisition rate may lead to the failure to record fast and small events. Second, the Ca2+ responses occur in small spatially constrained compartments (size around 1-lm length maximum) that are associated with dendritic spines and synapses. Owing to the complex, spongiform morphology of an astrocyte, it is difficult to perform imaging at the proper/ideal location. The temporally and spatially intimate relationship between synapses and their activity must be taken into consideration as they dictate the location and timing of the glial response to the minimal release of glutamate. As a whole, it is crucial that the acquisition be performed at a confirmed synaptic site using rapid acquisition rates (e.g. using the line scan mode of a confocal) (Panatier et al., 2011). Additional technical details can be found in (Panatier and Robitaille, 2013). In addition to their roles in the hippocampus, astrocytic mGluR5s also play crucial roles in the detection of synaptic transmission in the thalamus of young rats (Parri et al., 2010), in the nucleus accumbens (D’Ascenzo et al., 2007) and cortex of adult mice (Wang et al., 2006). Nedergaard and coworkers observed robust astrocytic Ca2+ events in response to in vivo tACPD application by iontophoresis in layer 2 of the barrel cortex in adult mice (Wang et al., 2006). More importantly, MPEP-sensitive astrocytic Ca2+ events were evoked by whisker stimulation in a frequency-dependent manner (Wang et al., 2006). However, in a more recent study, the same group failed to induce Ca2+ elevation in astrocytes of adult mice in vivo. These studies used a less well-controlled application of agonists co-injected with Alexa 488. More importantly, the magnification used to perform Ca2+ imaging was lower (Sun et al., 2013), implying that activity within the astrocytic processes where mGluR5 are preferentially located could have easily been missed. As a whole, data from Wang and collaborators are more supportive of the presence and expression of mGluR5s by cortical astrocytes in adults. However, their implication in the activation of astrocytes is in need of a systematic and careful analysis.
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Astrocytes are characterized by the presence of stubby main processes and dense ramifications of thinner processes with an irregular shape, giving them a
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spongiform morphology (Bushong et al., 2002, 2004). In the hippocampus (Bushong et al., 2002) and the cortex (Halassa et al., 2007) a single astrocyte has its own, non-overlapping territory, named the astrocytic domain. It has been estimated that each astrocyte could contact around 100,000 synapses (Bushong et al., 2002), and 4–8 neurons, encompassing 300–600 dendrites (Halassa et al., 2007). Hence, similarly to neurons, there are a large number of synapses that a given astrocyte is associated with. Accordingly, from a morphological to a functional point of view, astrocytes are well positioned to regulate synaptic transmission from a single synapse level (Panatier et al., 2011) to a neuronal network level (Araque et al., 2014). There are many neuronal and synaptic mechanisms that are regulated by astrocytes as a consequence of their synaptic activation. A number of these mechanisms are regulated by the activation of the glial mGluR5s. For instance, astrocytes detect synaptic transmission occurring at the single synapse level induced by minimal synaptic stimulation in the juvenile CA1 hippocampus (Panatier et al., 2011). Importantly, because of the electrophysiological tools and protocols used, only a single fiber was stimulated, leading to the activation of 1 to 2–3 synapses (Panatier et al., 2011).
Following this detection through mGluR5s, astrocytes release purines, which act on presynaptic adenosine A2A receptors to up-regulate basal synaptic transmission (Fig. 1, left). It is noteworthy that CA3–CA1 hippocampal synapses are tonically inhibited by astrocytic purines through the activation of another type of adenosine receptor, the presynaptic A1 receptor (Pascual et al., 2005). This interaction between A1R mediated tonic suppression of synaptic transmission and A2A-dependent up-regulation of synaptic transmission provides a mechanism through which synapses may be depressed or potentiated as a function of the activity in the network. In addition to purines there is also evidence that astrocytes release glutamate following stronger mGluR5 activation probably in a non-vesicular mode, as suggested by the absence of effect of tetanus toxin (Fig. 1, right) (Pasti et al., 1997; Angulo et al., 2004; Fellin et al., 2004). These authors reported that application of mGluR agonists induced the release of glutamate from astrocytes that in turn activates neighboring neuronal extrasynaptic glutamatergic N-methyl-D-aspartate (NMDA) receptors (Fig. 1, right) which induces an increase of neuronal Ca2+ (Pasti et al., 1997; Angulo et al., 2004; Fellin et al., 2004). Interestingly, the astrocytic glutamate activates specifically extrasynaptic
Fig. 1. Model of mGluR5-dependant neuronal-astrocyte interaction at hippocampal CA3–CA1 synapses. (Left) At the single synapse level, glutamate released after the arrival of a single action potential (1) activates astrocytic mGluR5s (2), leading to a local increase of Ca2+ in the astrocytic process (3). Subsequently Ca2+ triggers purine release. Once released, purines activate presynaptic A2A receptors (4), inducing an upregulation of the efficacy of transmission (5). (Right) During a sustained synaptic activity (1), glutamate activates astrocytic mGluR5s (2) leading to a large increase of Ca2+ (3) that propagates over a large portion of the astrocyte. Under this condition, glutamate is released from the astrocyte, activating extrasynaptic NMDARs (4). This release of glutamate from astrocyte controls neuronal excitability and synchrony (5). Please cite this article in press as: Panatier A, Robitaille R. Astrocytic mglur5s and the tripartite synapse. neuroscience (2015), http://dx.doi.org/ 10.1016/j.neuroscience.2015.03.063
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NMDA receptors (with NR2B subunit) in 2–4 neighboring neurons (Fellin et al., 2004). These NR2B-mediatied currents are called slow inward currents (SICs) (Fellin et al., 2004), or slow transient currents (STCs) (Angulo et al., 2004). They can either occur spontaneously at a very low frequency (Angulo et al., 2004; Fellin et al., 2004), or be evoked by Schaffer collateral stimulations (Fellin et al., 2004). Interestingly, these SICs could be synchronized within a time window of 100 ms in recorded neuronal pairs. Using this process, astrocytes are able to regulate neuronal excitability and synchrony (Angulo et al., 2004; Fellin et al., 2004). Importantly, a similar process has been suggested in the nucleus accumbens (D’Ascenzo et al., 2007).
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The localization and density of mGluR5s will influence the integration of synaptic transmission by astrocytes and, thus, will be dependent on the level of synaptic activation. As with other receptors in the brain, mGluR5 expression is regulated during development. To better understand the role of mGluR5s it is important to not consider only its expression but its localization, properties and functions must also be taken into account. Furthermore, the space and time between synapses and their activity are fundamental. Thus, it is crucial to adapt the time and space domains of the experimental paradigm to fit the kinetics and properties of the astrocytes in a given physiological condition. Astrocytic mGluR5s are playing fundamental roles in the detection of synaptic signals and thus, in turn, in the regulation of synaptic transmission. This has been shown particularly at hippocampal CA3–CA1 synapses. Major advances in the roles of mGluR5s will rely upon a better understanding of the molecular machinery in particular the mGluR5s scaffolding at synapses and their organization along processes in association with the internal stores of Ca2+.
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Acknowledgments—We would like to thank Dr Clare Reynell for her comments on the manuscript. This work was supported by grants from Marie Curie FP7 (FP7-PEOPLE-2010-RG) and CNRS to AP. RR is a recipient of grants from the Canadian Institutes for Health research to (MOP-14137 and MOP111070), a Leader Opportunity Fund from the Canadian Foundation of Innovation and an infrastructure grant from Fonds de Recherche du Que´bec - Sante´ (FRQ - Sante´) to the GRSNC (Groupe de recherche sur le syste`me nerveux central).
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(Accepted 26 March 2015) (Available online xxxx)
Please cite this article in press as: Panatier A, Robitaille R. Astrocytic mglur5s and the tripartite synapse. neuroscience (2015), http://dx.doi.org/ 10.1016/j.neuroscience.2015.03.063
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