Genetically Encoded Calcium Indicators and Astrocyte Calcium Microdomains

Abstract

The discovery of intracellular Ca²⁺ signals within astrocytes has changed our view of how these ubiquitous cells contribute to brain function. Classically thought merely to serve supportive functions, astrocytes are increasingly thought to respond to, and regulate, neurons. The use of organic Ca²⁺ indicator dyes such as Fluo-4 and Fura-2 has proved instrumental in the study of astrocyte physiology. However, progress has recently been accelerated by the use of cytosolic and membrane targeted genetically encoded calcium indicators (GECIs). Herein, we review these recent findings, discuss why studying astrocyte Ca²⁺ signals is important to understand brain function, and summarize work that led to the discovery of TRPA1 channel-mediated near-membrane Ca²⁺ signals in astrocytes and their indirect neuromodulatory roles at inhibitory synapses in the CA1 stratum radiatum region of the hippocampus. We suggest that the use of membrane-targeted and cytosolic GECIs holds great promise to explore the diversity of Ca²⁺ signals within single astrocytes and also to study diversity of function for astrocytes in different parts of the brain.

Keywords

GECI GCaMP3 GCaMP5G astrocyte process branchlet TRPA1 GAT-3 calcium leaflet endfoot branch

Astrocytes are found throughout the brain and their proximity to neurons has been known for over a century (DeFelipe 2009; Kettenmann and Verkhratsky 2008). Astrocyte density in the brain has increased dramatically through evolution (Nedergaard and others 2003). It is now well established that astrocytes serve vital support roles including buffering of K⁺ around neurons, regulation of blood flow, clearance of neurotransmitters from synapses, as well as providing nutrients and oxygen (Attwell and others 2010; Iadecola and Nedergaard 2007; Kofuji and Newman 2004; Magistretti 2006). In addition to these varied and vital supportive roles, increasing evidence suggests that astrocytes respond to, and regulate, neuronal function (Araque and others 2001; Halassa and Haydon 2010; Haydon 2001). In particular, attention has been focused on Ca²⁺ signals within astrocytes as a basis to explore, understand, and interrogate their roles within neuronal circuits. The strong focus on Ca²⁺ is based on the realization that Ca²⁺ is a ubiquitous second messenger within diverse cell types (Clapham 2007), prompting exploration of its roles within astrocytes.

Why We Need to Study Astrocyte Intracellular Ca²⁺ Signals ([Ca²⁺]_i)

The possibility that astrocytes may contribute to information processing in neuronal circuits was thoughtfully enunciated 20 years ago (Smith 1992), soon after Ca²⁺ signals were discovered within astrocytes (Charles and others 1991; Cornell-Bell and others 1990; Dani and others 1992). Astrocytes do not display propagated electrical signals analogous to action potentials (APs) in neurons and from an electrophysiological perspective appear silent. Their resting membrane potential rarely deviates by much from the K⁺ equilibrium potential (Mishima and Hirase 2010; Mishima and others 2007). However, astrocytes frequently display spontaneous intracellular Ca²⁺ concentration ([Ca²⁺]_i) elevations that can reach the micromolar range at peak height and are thus easily within the range of [Ca²⁺]_i known to affect cell physiology. Astrocyte [Ca²⁺]_i signals can also be evoked by activation of natively expressed G-protein coupled receptors (GPCRs) using exogenous agonists or by evoking neurotransmitter release from neuronal synapses (Agulhon and others 2008; Halassa and Haydon 2010). In some circumstances, most notably in vitro, astrocyte [Ca²⁺]_i signals may spread to entire astrocytes and even propagate to neighboring astrocytes as [Ca²⁺]_i waves (Bowser and Khakh 2007b; Cornell-Bell and others 1990; Dani and others 1992; Koizumi 2010; Scemes 2000). In view of these findings, astrocyte [Ca²⁺]_i signals have increasingly been considered to represent a form of excitability that may be caused by, control, trigger, or otherwise be correlated with alterations in neuronal function. Thus, it has been shown that astrocytes respond to neuronal signals in a process called neuron-to-astrocyte signaling and that astrocytes also regulate neuronal function via a variety of active and supportive mechanisms in a process called astrocyte-to-neuron signaling (Agulhon and others 2008; Halassa and Haydon 2010).

The last few years have witnessed a growth of interest in both astrocyte-to-neuron and neuron-to-astrocyte signaling, heralding the emergence of an exciting research area within neuroscience. In these settings, attention has focused on [Ca²⁺]_i dynamics as a form of encoded signal. In particular, in parallel with reports of astrocyte-to-neuron signaling via intercellular connections (Nedergaard 1994), it was shown that [Ca²⁺]_i increases cause release of glutamate from astrocytes that in turn affects neurons (Parpura and others 1994; Pasti and others 1997). Subsequent studies provided mounting evidence to suggest that gliotransmitters lead to changes in synapses (Lee and others 2007; Perea and others 2009) and behavior (Gourine and others 2010; Halassa and others 2009) via astrocyte-to-neuron signaling. However, despite impressive strides, the settings under which brain astrocyte-to-neuron signaling occurs are still not completely clear and this remains an important and ongoing task for the field. In particular, the relative contributions of vesicle- and channel-mediated release of gliotransmitters is unclear in many brain areas, although recent studies suggest that both mechanisms exist and that both should be considered on a case-by-case basis (Halassa and others 2009; Lee and others 2010; Pascual and others 2005; Woo and others 2012).

Several recent articles and reviews have considered astrocyte-to-neuron signaling in great depth, offering views and interpretations that both support and refute the existence, importance, and prevalence of this mechanism for brain function (Agulhon and others 2008; Halassa and Haydon 2010; Nedergaard and Verkhratsky 2012). We do not dwell on these issues here, in light of the breadth of these past undertakings.

We offer the parsimonious view that astrocytes may mediate diverse types of responses in different brain nuclei and under different cellular settings and that there may not be a single binary signal that astrocytes use in a way that is comparable to the use of APs by neurons. Indeed, this more nuanced and graded (as opposed to all-or-none) view of astrocyte [Ca²⁺]_i signals seems consistent with the physiology of astrocytes, which display tremendous diversity of form and function in different regions of the brain (Oberheim and others 2012; Sun and Jakobs 2011) and, of course, with the exception of endfeet, lack polarized specializations akin to axons and dendrites. Astrocytes also undergo reactive gliosis, which itself is a remarkably heterogeneous response leading to changes in gene expression in a context-specific manner (Sofroniew 2009; Zamanian and others 2012). In fact, the latest transcriptome studies show that different inflammatory mediators change astrocyte gene expression in distinct ways and produce strong changes in GPCRs that mobilize Ca²⁺ from intracellular stores (Hamby and others 2012). This speaks to the possibility that a given population of astrocytes may produce varied functional responses depending on the degree to which they are reactive. Ca²⁺ mediates myriad responses within cells (Clapham 2007), making it likely that its roles in astrocytes will also be equally diverse and varied. Finally, a pioneering study showed clear morphological differences in astrocytes even within subareas of the dentate gyrus, providing strong evidence for heterogeneity (Kosaka and Hama 1986). With these considerations in mind, it seems unlikely that a single stereotyped response to [Ca²⁺]_i exists in all astrocytes—simply because all astrocytes are not the same and Ca²⁺ does multiple things in different settings. It seems reasonable that [Ca²⁺]_i will serve specific functions in distinct settings. Perhaps the key to understanding what these ubiquitous and endlessly fascinating cells do in the brain is to embrace their diversity of form and function to explore specific questions, within specific brain nuclei and under specific experimental settings.

The Need for Improved Methods to Study Diverse Astrocyte Ca²⁺ Signals

To fully understand the physiology of astrocyte [Ca²⁺]_i signals, one needs methods to selectively monitor and evoke [Ca²⁺]_i signals within genetically specified astrocytes. Herein, we focus on recent work from our group on the use of genetically encode Ca²⁺ indicators (GECIs) to image [Ca²⁺]_i within astrocytes and how the use of membrane-tethered GECIs allowed us to discover a novel form of near membrane [Ca²⁺]_i signal in hippocampal astrocytes. The physiology of this [Ca²⁺]_i signal is just beginning to be explored. We do not discuss ways to evoke astrocyte [Ca²⁺]_i signals but refer the readers to general approaches developed to elevate [Ca²⁺]_i using modified GPCRs (Agulhon and others 2010; Conklin and others 2008) and optogenetics (Zhang and others 2011), as well as a specific method that has been shown to work beautifully for astrocytes (Li and others 2012). We restrict our focus to monitoring [Ca²⁺]_i signals within astrocytes, which we believe is the first step needed before we can devise the right strategies to trigger physiologically relevant [Ca²⁺]_i signals and explore their causative roles in circuits.

The development and use of [Ca²⁺]_i imaging techniques has been central to explore the physiology of astrocytes and has led to an explosion in our understanding of these cells. Astrocyte [Ca²⁺]_i signaling has been shown to occur in vitro within astrocyte cultures (Bowser and Khakh 2007a; Charles and others 1991; Cornell-Bell and others 1990; Dani and others 1992; Nedergaard 1994; Parpura and others 1994), in acute slice preparations (Agulhon and others 2010; Bacci and others 1999; Bowser and Khakh 2004; Fellin and others 2004; Fiacco and McCarthy 2004; Fiacco and others 2007; Nett and others 2002; Parri and Crunelli 2001; Pasti and others 1997; Perea and Araque 2007; Porter and McCarthy 1995a; Porter and McCarthy 1995b; Porter and McCarthy 1996; Sasaki and others 2011a; Sasaki and others 2011b; Shigetomi and others 2008; Shigetomi and others 2011), in vivo for anesthetized animals (Hirase and others 2004; Hoogland and others 2009; Navarrete and others 2012; Petzold and others 2008; Sasaki and others 2011a; Schummers and others 2008; Takata and others 2011; Thrane and others 2011; Wang and others 2006), in freely behaving animals (Nimmerjahn and others 2009), and within astrocytes from humans soon after biopsy removal of brain tissue (Oberheim and others 2009). Because Ca²⁺ is a ubiquitous second messenger, these findings draw attention to [Ca²⁺]_i signaling as a prevalent feature of astrocytes and raise important questions about the contribution of [Ca²⁺]_i signaling to astrocyte interactions with other cells. These include other types of astrocytes, neurons, microglia, NG2 cells, and blood vessels.

The need for improved methods to study astrocyte [Ca²⁺]_i signals is not driven by a technical quest but rather by knowledge of existing biology showing distinct types of [Ca²⁺]_i signals that differ in their spatial extent and in their duration. One recent study is of particular note. Nimmerjahn and others reported three different types of Ca²⁺ signals in Bergmann glia, which are specialized astrocytes in the cerebellum (Nimmerjahn and others 2009). By imaging [Ca²⁺]_i signals from awake and freely moving animals, they found evidence for “Ca²⁺ flares” that extend to more than 100 cells and occur during locomotion, “Ca²⁺ bursts” that expand radially as waves encompassing ~40 cells, and “Ca²⁺ sparklets,” which are local subcellular [Ca²⁺]_i signals partly independent of neuronal activity (Nimmerjahn and others 2009). These studies provide elegant proof that astrocyte [Ca²⁺]_i signals are a diverse group of events and that the physiological roles of each type will need to be explored in depth before we can surmise their physiological roles for the function of neural circuits.

To study astrocyte [Ca²⁺]_i dynamics, bulk loading of membrane-permeable organic Ca²⁺ indicator dyes has been extensively used because of the ease and relative specificity of the procedure for loading astrocytes in brain slices (Porter and McCarthy 1995a; Porter and McCarthy 1995b). This method remains powerful for studying [Ca²⁺]_i signals in the somata and main thick branches of astrocytes (see Box 1 for a note on the nomenclature we use in this review). However, these techniques suffer from severe limitations. The mechanism of astrocyte-specific labeling is unclear and likely represents a “plumbing” effect rather than a cell-surface protein-dependent uptake mechanism. As such, cell labeling can be quite uneven. Most important, it fails to report on finer branchlets perhaps because of the high-background noise within the neuropil and/or inadequate loading of Ca²⁺ indicator dyes into these small compartments. Indeed, quantitative Sholl analysis of astrocyte morphology suggests that as much as 90% of an astrocyte’s area was not sampled by bulk-loading methods (Fig. 1) (Reeves and others 2011). This is an important realization because astrocytes interact with neurons and blood vessels via their fine extensions and endfeet, respectively (Bergersen and others 2012; Bushong and others 2002; Mishchenko and others 2010; Ventura and Harris 1999). Electron microscopic reconstruction of astrocyte processes and endfeet revealed an extraordinarily complex network of small filaments (Mathiisen and others 2010). Therefore, it is unlikely that the full extent of astrocyte [Ca²⁺]_i signals has been studied in domains that are considered to be the sites of interaction with other cells (Reeves and others 2011). It seems likely that this disconnect between where astrocyte [Ca²⁺]_i signals have been measured (i.e., the soma) and where they underlie physiological responses (at the fine branchlets and leaflets; Box 1) has contributed to an incomplete picture and perhaps even led to the current controversy regarding the role of astrocytes in neuronal circuits (Agulhon and others 2008). In light of this, measuring [Ca²⁺]_i signals with improved resolution, at all relevant cellular substructures and within genetically defined populations of astrocytes, is an important and worthy goal.

Loading of membrane-impermeable organic Ca²⁺ indicator dyes via glass patch pipettes has also been used to measure astrocyte [Ca²⁺]_i signals (Di Castro and others 2011; Fiacco and others 2004; Fiacco and others 2007; Grosche and others 1999; Nett and others 2002; Panatier and others 2011). Direct pipette filling leads to fuller, and more target cell–specific, labeling of processes than bulk loading, although long, thin processes may remain unlabeled. A pioneering study was conducted by Grosche and others for Bergmann glia in the cerebellum (Grosche and others 1999). They found intracellular store-mediated microdomain [Ca²⁺]_i signals as a result of repetitive stimulation of parallel fibers. More recently, Panatier and others (2011) used a similar approach to dialyze organic Ca²⁺ indicator dyes into hippocampal astrocytes in brain slices and found that glutamate released from neurons evoked by single action potentials could evoke compartmentalized Ca²⁺ signals within astrocyte processes. Similarly, Di Castro and others (2011) found two types of localized Ca²⁺ signals in hippocampal astrocytes in the molecular layer of the dentate gyrus, which they termed focal and expanded Ca²⁺ signals. Focal signals were action potential-independent, confined signals with a size of ~4.2 µm and duration of ~700 ms. In contrast, expanded signals were larger (~12.9 µm), lasted longer (~3.2 s) and were driven by action potentials. Overall, Panatier and others and Di Castro and others developed on the work of Grosche and others and found evidence for local [Ca²⁺]_i signals within astrocyte branches that they concluded were important for regulating basal synaptic transmission in a gliotransmitter-dependent manner.

Box 1. On Astrocyte Morphology Nomenclature: Branches, Branchlets, and Leaflets

It has been known since the time of Cajal that astrocytes are highly branched and ramified cells (DeFelipe 2009; Kettenmann and others 2008), a feature that has been repeatedly demonstrated in subsequent anatomical studies, most notably for human astrocytes (Oberheim and others 2009). However, as discussed in the main text, most [Ca²⁺]_i imaging studies have focused on astrocyte somata, which are easy to identify and evaluate. As optical methods have improved, focus has shifted to the study of astrocyte processes located at variable distances away from the soma (Agulhon and others 2010; Di Castro and others 2011; Grosche and others 1999; Reeves and others 2011) and in one case to the study of “enlargements” on astrocyte processes (Panatier and others 2011). In such studies, the term astrocyte process has been used to discriminate the signals from those recorded in the somata, but it remains unclear exactly what this means in particular studies because astrocytes possess many, perhaps hundreds, of processes.

Defining what is meant by “astrocyte process” is important to understand and thoughtfully compare [Ca²⁺]_i signals between studies and also to make inferences about the relationship of [Ca²⁺]_i signals to synapses, which are contacted by the myriad terminal endings of astrocytes and seemingly not by thick processes (Bergersen and others 2012; Bushong and others 2002; Harris and others 2012; Mishchenko and others 2010; Ventura and Harris 1999; Witcher and others 2010).

We suggest that the term astrocyte process should evolve into several related terms that convey more meaningful information about the astrocyte subcompartment being studied within intact preparations such as brain slices and in vivo. We provide some working definitions below.

Branches are the major processes emanating from the astrocyte soma and the focus of recent attention (Di Castro and others 2011; Grosche and others 1999; Panatier and others 2011; Reeves and others 2011). These probably do not number more than 8 per astrocyte and display diameters on the 1 to 2 µm scale (Reeves and others 2011) and have also been called “stem processes” in the literature.

Branchlets are the finer secondary, tertiary, and higher order structures that emanate from branches. The precise number of these is presently unknown, but they display diameters on the sub–micrometer scale (Reeves and others 2011). Conventional diffraction limited light microscopy may not be ideal to study the finest branchlets satisfactorily, although they can be seen clearly as distinct from branches.

Leaflets are the very terminal extensions of astrocyte branchlets that contact synapses (Bergersen and others 2012; Bushong and others 2002; Harris and others 2012; Mishchenko and others 2010; Ventura and Harris 1999; Witcher and others 2010). The precise numbers of these per astrocyte is currently unknown, but based on electron microscopy studies they display dimensions on the tens of nanometer scale (Hama and others 2004). By our definition, leaflets would be the same structures as those variously termed “astrocyte lamellae,” “astrocyte sheets,” “veillike lamellae,” “peripheral astrocyte processes,” and “astrocyte fingers” (Derouiche and Frotscher 2001). Direct comparison of light microscopy with high-voltage electron microscopy shows that leaflets cannot be imaged with diffraction-limited light microscopy (Hama and others 2004). Moreover, in the dentate gyrus molecular layer leaflets have dimensions of tens of nanometers at sites of interaction with dendrites (Kosaka and others 1986), that is, they are far smaller than the micrometer scale thick branches that have been imaged by two-photon microscopy (Di Castro and others 2011).

Endfeet are specialized distal extensions of astrocytes that contact the vasculature. One expects that each astrocyte bears only one or two branches with enlarged endfeet. Endfeet appear to represent a truly polarized compartment of astrocytes with a well accepted function.

The more general term of astrocyte processes is still useful and could be used loosely, for example, to describe astrocytes in cell culture where they have lost their native morphology or when the location of a particular signal does not matter or encompasses all processes (e.g., “process retraction”).

We finish by adding that our suggested nomenclature is, of course, not presented as the final word on the matter. However, we hope it may serve as a basis for further discussion and hopefully the field will converge on definitions of astrocyte compartments that can be accepted and used as a basis to explore and study astrocyte physiology with a common and understood language. Our logic in suggesting better terminology is simple: if we want to determine how astrocytes contribute to neuronal circuits, then we must know how the signals being measured by different laboratories relate to each other from an anatomical perspective and also to the sites of interaction with synapses. This is currently unclear from the existing literature and deserves to be addressed.

Overall, patch-mediated dialysis of astrocytes with Ca²⁺ indicator dyes represents an improvement over the use of bulk loading; however, it is still far from a panacea and introduces several problems of its own. First, patch mediated dialysis of Ca²⁺ indicator dyes does not provide information on astrocyte branchlets and leaflets: Panatier and others and Di Castro and others studied processes that were only 5 to 20 µm from the soma and between 1.2 and 1.4 µm wide, that is, these main branches were far larger than the fine less than hundreds of nanometer scale branchlets and leaflets that are widely considered to be the sites of interaction with synapses based on electron microscopy studies (Bergersen and others 2012; Bushong and others 2002; Harris and Weinberg 2012; Kosaka and Hama 1986; Mishchenko and others 2010; Ventura and Harris 1999; Witcher and others 2010). Second, Di Castro and others used quite high amounts of Ca²⁺ indicator dye (0.2 mM), leaving open the possibility that this procedure altered [Ca²⁺]_i buffering. Third, by definition, the act of dialyzing cells not only introduces the exogenous dye into them but also washes out intracellular components from within. This is a concern for all such experiments, but is particularly relevant to the study of astrocytes, which are known to couple extensively via gap junctions into a syncytium. Indeed, astrocytes in the molecular layer of the dentate gyrus form sponge-like conglomerates via fine leaflets (Kosaka and Hama 1986). One wonders therefore if the act of patching and dialyzing single astrocytes actually alters their functions in a way that is more perturbing than for other more weakly coupled cells. It is noteworthy that patch pipette mediated dialysis is often used as a way to perturb astrocytes (e.g., to wash in toxins and Ca²⁺ buffers), meaning that coincidental wash out must happen when using the very same method to deliver Ca²⁺ indicator dyes, which ideally should be innocuous. McCarthy and colleagues have advocated brief dialysis times with high dye concentrations (Agulhon and others 2010; Nett and others 2002), which of course will help, but even this does not remedy the problem. Indeed, this suggestion is a persuasive argument that the problem of dialysis is real and should not be ignored (Nett and others 2002). Irrespectively, the work by Panatier and others and Di Castro and others does suggest that astrocytes may signal locally to neurons, and therefore represents a call for improved methods to study local [Ca²⁺]_i signals noninvasively in astrocytes using methods that remedy some or all of the aforementioned shortcomings of organic Ca²⁺ indicator dyes.

Genetically Encoded Calcium Indicators and Fluorescence Imaging

Genetically encoded calcium indicators circumvent many of the problems presented by small molecule dyes. GECIs are typically composed of a Ca²⁺-binding domain and one to two fluorescent proteins (FPs), the properties of which are modulated in a Ca²⁺-dependent fashion, leading to a change in fluorescence excitation/emission wavelengths, intensities, or Förster resonance energy transfer. Fluorescent proteins, such as the widely used GFP, are autocatalytic, requiring no exogenous cofactors. As such, they, and sensors derived from them, may be encoded in single polypeptide chains and targeted via cell type–specific promoters to particular populations of cells and/or via designed fusion proteins to specific subcellular compartments. Expressed protein probes can be repeatedly imaged over weeks and months, allowing visualization of long-term processes such as development and learning. Delivery of transgene from viral infection or in utero electroporation can target large volumes of cells, and the labeling that results from endogenous expression is far more uniform than from small molecule injection. Labeling density may be carefully titrated depending on the application, from all cells in a region to sparse, individually resolvable cells. In vivo electroporation (Boutin and others 2008) is a recent technique facilitating the uniform loading of single cells with genetically encoded reporters.

The most relevant GECI properties for neuroscience applications include signal-to-noise ratio (SNR), Ca²⁺ affinity, rise and decay kinetics, dynamic range, photostability, compatibility with long-term imaging, and targeting to specified locations and fusion proteins. The most optimized GECI scaffold is the GCaMP scaffold, initially engineered in 2001 (Nakai and others 2001), with numerous subsequent improvements (e.g., GCaMP1.6, GCaMP2, GCaMP3, GCaMP5, and GCaMP-HS) that have been recently reviewed (Tian and others 2012). The determination of the high-resolution structures of the Ca²⁺-bound and Ca²⁺-unbound forms of the sensor (Akerboom and others 2009; Wang and others 2008) has significantly improved efforts to improve its properties, as rational design and focused-library screening have largely validated the relevance of the crystal structures to in situ performance. Current GCaMP variants can reliably detect single APs in acute mouse brain slice, and bursts of ~3 APs in vivo (GCaMP3, Tian and others 2009; GCaMP5, Akerboom and others 2012). Imaging-defined populations of ~100 to 200 neurons over weeks to months with GCaMP3 has revealed circuit-level changes in behavioral task-dependent neuronal activity (Huber and others 2012).

Astrocyte imaging places somewhat different restrictions on GECI performance criteria than for those for optimal imaging in neurons. For instance, neuronal membranes are highly electrically excitable, with fast rise and decay rates of the voltage signal controlled by the action potential machinery. Voltage-gated Ca²⁺ channels opened by APs result in large, fairly rapid Ca²⁺ fluxes propagated through dendrites and the soma. Astrocytes are not electrically excitable, except at their specialized endfeet (see Box 1), and as such Ca²⁺ must enter through ligand-gated channels (and gap junctions to neighboring astrocytes), or be released from intracellular stores. These lower levels of [Ca²⁺] flux demand either higher-affinity GECIs or the targeting of the GECIs to the source of the Ca²⁺ entry. Given the absence of the AP excitation mechanism in astrocytes, Ca²⁺ fluxes are likely to be slower in both rise and decay than in neurons, lowering the bar for GECI kinetics. Indeed, even the signals termed fast typically last >0.5 seconds within astrocytes.

Obviously, there are different demands for in vitro and in vivo imaging, as well. In vivo imaging dramatically degrades SNR, particularly as imaging depth increases. Two-photon imaging can bleach probes at much higher rates than one-photon illumination, particularly for targeted, slowly diffusing fluorophores. Scattering, autofluorescence, hemodynamics, and animal-motion artifacts all significantly pollute imaging channels. As in vivo samples behave as light sinks, phototoxicity concerns require lower laser powers, which directly compete with probe fluorescence output. Furthermore, mammals typically have body temperatures of 34 to 37 °C, increasing the speed at which [Ca²⁺]_i signals propagate relative to room temperature that is often used for in vitro studies.

GCaMPs and the Lck Tag: Discovery of Spotty [Ca²⁺]_i Signals

We recently sought to develop a noninvasive method to measure [Ca²⁺]_i signals in astrocytes using GCaMP variants (Hires and others 2008; Tian and others 2009). In the past, we had enjoyed some success imaging Ca²⁺ near the inner mouth of an ion channel (Chaumont and others 2008; Richler and others 2008), and in light of this experience we focused on using membrane-targeted GECIs. There were also three reasons from an astrocyte perspective. First, work with astrocyte-neuron cocultures and total internal reflection fluorescence microscopy (TIRFM) has shown that astrocytes display near membrane [Ca²⁺]_i signals that were not observed with conventional wide field microscopy (Shigetomi and Khakh 2009; Shigetomi and others 2010b). However, TIRFM can only be used in cell culture and so another method was needed to explore near membrane [Ca²⁺]_i signals in astrocytes in brain slices and in vivo. Second, rapid switching between TIRFM and wide field microscopy revealed that many cytosolic [Ca²⁺]_i signals failed to elevate Ca²⁺ near the plasma membrane, implying that cytosolic signals could not be used as a surrogate measure of events near the plasma membrane. Third, we were cognizant of the fact that the sites of interaction between neurons and astrocytes were likely to be small tubular structures that by definition have high surface areas and low internal volumes (Hama and others 2004; Ventura and Harris 1999), implying that a membrane-targeted GECI may be better than a cytosolic one to image [Ca²⁺]_i signals within them. Based on these considerations, we generated Lck-GCaMP2 (Shigetomi and others 2010b), which we improved further to generate Lck-GCaMP3 (Shigetomi and others 2010a) (Fig. 2). In this context, “Lck” refers to a 26 amino acid peptide from the Lck tyrosine kinase that has been shown to be a strong membrane tether (Benediktsson and others 2005; Zlatkine and others 1997). Consistent with our expectations, we found that Lck-GCaMP2 was ~10 fold better than cytosolic GCaMP2 for measuring near membrane [Ca²⁺]_i signals, that it was evenly and strongly distributed throughout the plasma membrane, and that it diffused as expected within the plasma membrane, implying there were no gross barriers to hinder its diffusion into fine branchlets (Shigetomi and others 2010b). On the other hand, there do appear to be significant barriers to the diffusion of cytosolic dyes into branchlets via the cytosolic bulk (Reeves and others 2011). Lck-GCaMP3 shares all these features of Lck-GCaMP2, with the additional boon that it displays a approximately twofold to threefold fold better SNR because of three designer point mutations (Shigetomi and others 2010a). Most recently, we made Lck-GCaMP5G and have shown that it improves the peak response size (dF/F) for astrocyte [Ca²⁺]_i signals by a further approximately twofold over Lck-GCaMP3 because it carries three new point mutations (Akerboom and others 2012). However, Lck-GCaMP5G tends to be dimmer than Lck-GCaMP3 (i.e., resting F is lower) and does not reveal new signals that were undetected by Lck-GCaMP3 (Akerboom and others 2012). Thus overall, Lck-GCaMP3 and Lck-GCaMP5G can be used to study near membrane [Ca²⁺]_i signals in astrocytes (Fig. 2). Our data suggest that membrane-tethered GECIs are far better than cytosolic dyes because they place the GECI in the membrane of fine processes where Ca²⁺ fluxes are likely to be greatest. Our use of a membrane tether was reminiscent of the use of other designer tags to place GECIs near sites of interest. For example GECIs have been targeted to neurotransmitter release sites in neurons with great success (Akerboom and others 2012; Dreosti and Lagnado 2011; Dreosti and others 2009). Table 1 summarizes the GECIs that have been used for astrocyte studies and provides details on how to obtain the plasmids.

Table 1.

Summary of the Available GECI Plasmids and Mice to Study Astrocyte Calcium Signals

GECI	Database ID	Made by	Reference
Plasmid tools
Lck-GCaMP2	Addgene # 24794	Khakh Lab	(Shigetomi and others 2010b)
GCaMP3	Addgene # 22692	Looger Lab	(Tian and others 2009)
Lck-GCaMP3	Addgene # 26974	Khakh Lab	(Shigetomi and others 2010a; Shigetomi and others 2012)
GCaMP5G	Addgene # 31788	Looger Lab	(Akerboom and others 2012)
Lck-GCaMP5G	Addgene # 34924	Khakh and Looger Labs	(Akerboom and others 2012)
In vivo transgenic mouse tools
S100β-YC 3.60	—	Russell Lab	(Atkin and others 2009)
ROSA26-CAG-floxedSTOP-GCaMP3	JAX Mice: Ai38, #014538	Chen Lab	(Zariwala and others 2012)

Note: GECI = genetically encoded Ca2+ indicator; “—” indicates that it is currently unknown if these reagents are available openly. The original GCaMP2 plasmid was developed by Kotlikoff Lab (Tallini and others 2006) and subsequently modified by mutagenesis to generate GCaMP3 and GCaMP5G (sometimes abbreviated simply as GCaMP5) (Akerboom and others 2012; Tian and others 2009). All three GCaMPs have been tagged with the strong membrane tether called Lck (Akerboom and others 2012; Shigetomi and others 2010a; Shigetomi and others 2010b; Shigetomi and others 2011). All the plasmids listed in this table are freely available from Addgene and can be ordered online using the listed Addgene IDs (http://www.addgene.org). We refer the readers to the main text for the advantages of each type of GECI for imaging Ca2+ signals in astrocytes and their branches and branchlets. In brief, GCaMP5G displays the largest dF/F, but has lower F than GCaMP3. Both GCaMP5G and GCaMP3 detect an equivalent number of spotty Ca²⁺ signals in astrocytes, and we conclude that both are about equally useful in this regard for detecting slow astrocyte signals, although GCaMP5G is significantly better than GCaMP3 for detecting fast neuronal signals (Akerboom and others 2012).

Introduction of Lck-GCaMP2, 3, and 5G into cultured hippocampal astrocytes revealed a novel type of microdomain Ca²⁺ signal, which we call “spotty Ca²⁺ signals” (Shigetomi and others 2010a; Shigetomi and others 2010b; Shigetomi and others 2011). Spotty Ca²⁺ signals (measured at room temperature) occurred randomly and frequently at numerous spots with an average size of ~5 µm and a duration of ~3.7 seconds (Fig. 3). Spotty Ca²⁺ signals are due to Ca²⁺ entry from the extracellular space and are resistant to strategies that block or deplete Ca²⁺ release from intracellular Ca²⁺ stores (Shigetomi and others 2011). Interestingly, spotty Ca²⁺ signals occur without any overt stimulation, and they are not affected by activation of a variety of GPCRs with exogenous agonists (Shigetomi and others 2011). Taken together, these findings suggest that the mechanisms underlying spotty Ca²⁺ signals are different from those for the more classical GPCR-mediated [Ca²⁺]_i signals frequently studied within astrocytes (Fiacco and others 2009). Moreover, the spotty Ca²⁺ signals are not caused by the expression of Lck-GCaMPs because similar signals could be observed using organic Ca²⁺ indicator dyes and TIRFM (Shigetomi and Khakh 2009; Shigetomi and others 2010b). However, the spotty Ca²⁺ signals are much easier to observe with Lck-GCaMPs and could also be studied with conventional wide field and multiphoton microscopy. When using wide field imaging, spotty Ca²⁺ signals are easily and reliably detected by membrane-targeted GECIs but infrequently using cytosolic GECIs, implying that membrane targeted GECIs are closer to the transmembrane source of local Ca²⁺ and that diffusion of the cytosolic GECIs may be suboptimal to detect near membrane events (Shigetomi and Khakh 2009; Shigetomi and others 2010a; Shigetomi and others 2010b; Shigetomi and others 2011).

Diversity of Ca²⁺ Signals in Astrocytes

Astrocytes constitute a diverse “cell type,” the breadth of which is only now beginning to be understood. Long thought of as a homogeneous population of “glue” or “support” cells, astrocytes are now recognized to come in different flavors. The glial fibrillary acidic protein (GFAP), previously believed to be a universal astrocyte marker, is now acknowledged to label only a subset of astrocytes; the aldehyde dehydrogenase enzyme Aldh1L1 is the current best pan-astrocyte marker (Cahoy and others 2008).

Even within a given “type” of astrocyte, [Ca²⁺]_i signaling physiology may also be quite diverse. Although there are only a handful of available studies, the resting [Ca²⁺]_i level of astrocytes is thought to be ~100 nM (Kuchibhotla and others 2009). Because astrocytes express a large variety of GPCRs that mobilize Ca²⁺ from intracellular stores, fluctuations in the resting [Ca²⁺]_i level of astrocytes have been extensively studied in the somata during pharmacological and physiological activation of GPCRs (Fiacco and others 2009). Activation of GPCRs causes inositol 1,4,5-triphosphate (IP₃) production through phospholipase C (PLC), leading to opening of IP₃ receptor Ca²⁺ channels on the endoplasmic reticulum (ER) to release Ca²⁺ from intracellular stores. Interestingly, although there are three types of IP₃ receptor, only type 2 receptors (i.e., IP₃R2) are expressed predominantly within astrocytes from the cortex and hippocampus. Accordingly, IP₃R2 knockout mice do not show spontaneous [Ca²⁺]_i elevations and lack [Ca²⁺]_i signals evoked by GPCR activation in the somata of hippocampal and cortical astrocytes loaded with organic [Ca²⁺]_i indicator dyes (Navarrete and others 2012; Petravicz and others 2008; Takata and others 2011). Thus, the molecular basis of a well-studied form of astrocyte [Ca²⁺]_i signal is release from intracellular stores via type 2 IP3 receptors; the physiological roles of these [Ca²⁺]_i signals for neuron-astrocyte and astrocyte-neuron communication have been extensively reviewed (Fiacco and others 2009).

In contrast to the store-mediated signals, our findings with Lck-GCaMPs suggest that the spotty Ca²⁺ signals represent an additional [Ca²⁺]_i signal that is spatially restricted to near membrane regions, including within astrocytic processes (Shigetomi and others 2010b; Shigetomi and others 2011). Furthermore, pharmacological analyses show that spotty Ca²⁺ signals are distinct from global [Ca²⁺]_i signals (Shigetomi and others 2010b). This is of interest because empirical data show that distinct sources or types of [Ca²⁺]_i signal may serve distinct physiological roles within astrocytes. Thus, photolysis of caged Ca²⁺ within astrocytes is known to cause both slow inward currents (mediated by extrasynaptic NMDA receptors) and facilitation of excitatory postsynaptic currents (EPSCs) onto neurons (Fellin and others 2004; Perea and Araque 2007; Sasaki and others 2011a). In contrast, photolysis of caged-IP₃, which elevates [Ca²⁺]_i levels through release of intracellular stores, only facilitates EPSCs (Fiacco and McCarthy 2004). Moreover, two almost identical [Ca²⁺]_i signals evoke different forms of astrocyte single vesicle exocytosis in cell culture (Bowser and Khakh 2007a) and two almost identical [Ca²⁺]_i signals lead to distinct effects on pyramidal neurons in acute brain slices (Shigetomi and others 2008). Additionally, Gordon and others (Gordon and others 2009) reported that local [Ca²⁺]_i signals within astrocyte processes are related to functional interactions with hypothalamic neurons, whereas somatic astrocyte [Ca²⁺]_i signals are not always correlated with neuronal effects. Taken together, these data imply that the details, such as the source, location, and dynamics of [Ca²⁺]_i, are important and may dictate, or at least regulate, how astrocytes respond to [Ca²⁺]_i elevations in terms of their effects on neurons. More generally, as discussed earlier, astrocyte [Ca²⁺]_i signals do not appear to be all-or-none binary signals, like action potentials in neurons. With these considerations in mind, we set out to identify the molecular basis of the spotty Ca²⁺ signals measured with Lck-GCaMPs in astrocyte-neuron cocultures because we thought they may be physiologically relevant for astrocytes.

By combining a pharmacological screening approach with the use of siRNAs, we discovered that spotty Ca²⁺ signals in astrocyte-neuron cocultures were mediated by TRPA1 channels (Shigetomi and others 2011). As far as we know, the TRPA1-mediated [Ca²⁺]_i signaling pathway has not been previously reported in past astrocyte studies, which have focused largely on [Ca²⁺]_i elevations due to store-mediated Ca²⁺ release (Agulhon and others 2008; Agulhon and others 2010). The discovery of functional TRPA1 channels in astrocytes and their contribution to near membrane and basal [Ca²⁺]_i signals adds to the richness and diversity of [Ca²⁺]_i signals in astrocytes (Gordon and others 2009; Shigetomi and others 2008), which also includes Ca²⁺ influx via ionotropic receptors such as Ca²⁺ permeable AMPA receptors, NMDA receptors, and P2X receptors (Nedergaard and Verkhratsky 2012) as well as reverse activation of Na⁺/Ca²⁺ exchangers (Nedergaard and Verkhratsky 2012).

TRPA1 Channels Contribute to Basal Calcium Levels in Astrocytes

Transient receptor potential (TRP) channels are a large family of nonselective cation channels (Wu and others 2010) that are grouped into six subfamilies based on sequence homology: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPML (mucolipin), and TRPP (polycystin). As a family, TRP channels are opened by a diverse range of physiological and pathophysiological stimuli, functioning as thermosensors, mechanosensors, and chemosensors. Most TRP channels are permeable to Ca²⁺ and contribute transmembrane Ca²⁺ fluxes. Because of their multimodality and their Ca²⁺ permeability the functional properties, biophysical mechanisms and physiological roles of TRP channels represent a fascinating area of biology (Wu and others 2010).

A wide variety of TRP channels could potentially be expressed in cultured astrocytes based on mRNA and protein expression studies. The pioneering transcriptome analysis by Cahoy and others (Cahoy and others 2008) showed TRPC1-2 and TRPM2-7 mRNA expression within acutely dissociated and immunopanned astrocytes that most likely reflect astrocytes in vivo (Cahoy and others 2008). However, several TRP channels were not seen to be astrocyte enriched and were under (or within) the limits of detection, including TRPA1 (Cahoy and others 2008). Of the TRP channels, mRNAs of TRPM3 and TRPM7 were highly expressed in astrocytes, although the functional significance of these in astrocytes is currently unknown. In light of these careful transcriptome studies, we were surprised to find functional evidence for TRPA1 expression within astrocytes from astrocyte-neuron cocultures and within acute brain slices (Shigetomi and others 2011). There is no immediately satisfying explanation for this difference between our functional studies and past mRNA expression analysis. However, several lines of evidence suggest that astrocytes express functional TRPA1 channels in culture and in acute brain slices.

First, spotty Ca²⁺ signals in astrocytes were reduced by siRNAs against TRPA1 and were almost completely abolished by the specific TRPA1 blocker, HC 030031. Second, activation of TRPA1 by low concentrations of AITC (the pungent ingredient in wasabi) increased the number of spotty Ca²⁺ signals. Third, several known TRPA1 activators/agonists caused global Ca²⁺ increases in astrocytes. Fourth, AITC-evoked Ca²⁺ elevations were reduced by both siRNA against TRPA1 and HC 030031. Fifth, AITC-evoked currents in astrocytes were reduced by siRNA against TRPA1. Sixth, TRPA1 protein expression was detected by Western blotting. Seventh, blockade of TRPA1 reduced basal [Ca²⁺]_i levels in astrocytes within cultures and acute slices. Eighth, the effect of TRPA1 blockade on astrocytes in acute hippocampal slices was significantly reduced in TRPA1 deletion (Trpa1^−/−) mice.

We suggest low-level expression of TRPA1 mRNAs may be sufficient to account for the spotty Ca²⁺ signals. In accord, TRPA1 is a relatively high-conductance cation channel with high Ca²⁺ permeability (fractional Ca²⁺ current ~17%; Nilius and others 2011) and TRPA1 undergoes pore-dilation, which further increases Ca²⁺ permeability (Chen and others 2009). Additionally, TRPA1 channels are also regulated by Ca²⁺, both through channel desensitization and through the function of an intracellular EF-hand Ca²⁺-binding domain (Wu and others 2010). This potentially establishes complex feedback loops between TRPA1, Ca²⁺, and other channels. A further issue also merits mention. It is now clear that the act of culturing astrocytes changes their complement of mRNAs (Cahoy and others 2008; Foo and others 2011; Zamanian and others 2012) and so it is possible that the TRPA1 responses were affected by such changes in our astrocyte-neuron cocultures. However, this would not explain our data from acute brain slices. The Barres lab has made tremendous progress in developing a culture system that maintains purified astrocytes in a native in vivo like state (Foo and others 2011). This represents the state-of-the-art for work on pure astrocyte cultures, but a similar preparation for astrocyte-neuron cocultures does not currently exist, and our goals were to study astrocytes with neurons, as in the brain. On balance, our findings, and perhaps all data from astrocyte-neuron cocultures, should be interpreted with these considerations in mind (Foo and others 2011). One looks forward to further advances with cell culture methodology that preserve astrocytes with in vivo like properties when cocultured with neurons.

An important aspect of TRPA1-mediated spotty Ca²⁺ signals within astrocytes is that they contribute to basal cytosolic [Ca²⁺]_i levels. Thus, inhibition of TRPA1 channels significantly reduces basal [Ca²⁺]_i levels from ~120 to ~60 nM (Shigetomi and others 2011). It remains feasible that the spotty Ca²⁺ signals may also regulate astrocyte [Ca²⁺]_i levels locally within microdomains, but the available data instead suggest that they largely contribute to setting bulk intracellular basal [Ca²⁺]_i levels. An additional feature of the TRPA1-mediated spotty Ca²⁺ signals that set them apart from other astrocyte [Ca²⁺]_i signals is that we found no evidence to suggest that they were regulated by, or required, neuronal activity or GPCR activation (Shigetomi and others 2011). This lack of regulation by mechanisms that are known to regulate intracellular store–mediated [Ca²⁺]_i signals further suggests that the function of TRPA1 may be to regulate basal [Ca²⁺]_i levels rather than be regulated by neuronal function, that is, at this juncture it seems unlikely that TRPA1 channels contribute to neuron-to-astrocyte communication in a way directly analogous to GPCR-mediated signals (Agulhon and others 2008). Further experiments to prove or refute these hypotheses are certainly needed. Moreover, given that TRPA1 channels are multimodal receptors activated by diverse stimuli (Wu and others 2010), it will also be important to evaluate if astrocyte TRPA1 channels are regulated by other processes, such as blood flow (Attwell and others 2010), brain injury, and disease (Kremeyer and others 2010).

We would be remiss if we did not mention other TRP channels in astrocytes, although our focus by necessity is TRPA1. A variety of approaches for astrocytes from different parts of the brain have provided evidence for the expression of TRPC1, TRPC3, TRPC6, TRPV1, and TRPV4 channels within astrocytes with important functional roles (Akita and Okada 2011; Benfenati and others 2011; Beskina and others 2007; Butenko and others 2012; Golovina 2005; Grimaldi and others 2003; Huang and others 2010; Lanciotti and others 2012; Liu and others 2009; Malarkey and others 2008; Shirakawa and others 2010; Wu and others 2010). Broadly, this emerging data set speaks to the potential importance of the TRP channel family for astrocytes and calls for more detailed explorations of their pathophysiological roles in specific brain nuclei. TRPM3 and TRPM7 seem to be particularly interesting based on transcriptome analysis (Cahoy and others 2008; Lovatt and others 2007).

TRPA1 Channels in Astrocytes Indirectly Modulate Inhibitory Synapses

We explored the role of astrocyte TRPA1 channels on synaptic transmission in the stratum radiatum region of the CA1 area of the mouse hippocampus. Pharmacological blockade of TRPA1 channels decreased the amplitude of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) onto interneurons by ~20%, but did not affect mIPSCs onto pyramidal neurons (Shigetomi and others 2011). The effect of TRPA1 blockade on interneuron mIPSCs was mimicked by clamping astrocyte [Ca²⁺]_i to ~35 nM, but was unaffected when [Ca²⁺]_i was clamped close to its resting level at around ~120 nM, implying that basal [Ca²⁺]_i was important and that the act of patching astrocytes per se was not (Shigetomi and others 2011). Importantly, the effect of TRPA1 blockade on mIPSC amplitudes onto interneurons was occluded by prior astrocyte dialysis with BAPTA to strongly buffer [Ca²⁺]_i levels in the astrocyte syncytium, suggesting that TRPA1 blockade with HC 030031 acted via astrocytes and contributed to their basal [Ca²⁺]_i. Moreover, applications of HC 030031 did not affect mIPSCs in mice lacking TRPA1 channels, indicating that the drug acted on TRPA1 to produce its effect rather than some other mechanism (Shigetomi and others 2011). Taken together, these data provide strong evidence for the functional consequences of TRPA1 expression within astrocytes.

During the course of the aforementioned studies, it became clear that astrocyte dialysis with BAPTA or TRPA1 blockade also resulted in enhanced GABA_A receptor–mediated tonic currents in interneurons that were sensitive to the competitive antagonist bicuculline. This finding suggested elevated GABA levels in the extracellular space during BAPTA dialysis and TRPA1 blockade, which may cause the tonic currents and lead to decreases in the amplitude of mIPSCs through desensitization of synaptic GABA_A channels. Indeed, blocking GAT-3 GABA transporters, which are enriched within astrocytes, mimicked the effects of astrocyte BAPTA dialysis and TRPA1 blockade, and the effects were mutually occlusive, suggesting that BAPTA dialysis and TRPA1 blockade both converge on GAT-3 (Shigetomi and others 2011). We found no evidence that other GABA transporters were involved (i.e., GAT-1 and GAT-2). Our findings are consistent with the widely accepted view that GABA transporters regulate the spillover of GABA into extrasynaptic regions (Beenhakker and Huguenard 2010), and accordingly, we found robust expression of GAT-3 in both the somata and processes of astrocytes recalling past work showing that GAT-3 is the major type of astrocyte GABA transporter (Minelli and others 1995; Minelli and others 1996; Ribak and others 1996).

Taken together, our functional analyses in brain slices suggests that the major function of astrocyte TRPA1 channels is to regulate basal [Ca²⁺]_i levels, which are permissive for the proper functioning and/or surface expression of GAT-3 (Shigetomi and others 2011). Once TRPA1 is blocked or astrocyte [Ca²⁺]_i is buffered, GAT-3 function and/or surface expression is decreased, leading to elevated extracellular GABA levels and reduced mIPSCs onto interneurons due to desensitization of GABA_A receptors (Fig. 4). In this context, astrocyte [Ca²⁺]_i levels are important for astrocyte-neuron communication, but the mechanism appears to involve a classical homeostatic function of astrocytes, namely, clearance of neurotransmitter rather than Ca²⁺-dependent gliotransmission. Also, the effect of astrocyte [Ca²⁺]_i levels is indirect and neuromodulatory rather than directly stimulatory or inhibitory.

We noted earlier that Ca²⁺ entry through TRPA1 channels contributes to basal [Ca²⁺]_i levels in astrocytes, and we further found that Ca²⁺ entry also acted locally to regulate GAT-3 (Shigetomi and others 2011). Thus, we compared GAT-3-mediated responses after dialyzing astrocytes with BAPTA and EGTA, which differ in their association kinetics for Ca²⁺ (Soeller and Cannell 2004). Estimation of length constants with these buffers suggests that GAT-3 is located within about 250 nm of the source of Ca²⁺ that regulates it (i.e., TRPA1). This unexpected finding strongly implies the existence of localized [Ca²⁺]_i signaling in astrocytes, which may be particularly important for the physiology of fine astrocyte branches, branchlets, and leaflets. The finding that Ca²⁺ acts locally in astrocytes to regulate GAT-3 recalls work by Henneberger and others, which demonstrated the importance of nanodomain astrocyte [Ca²⁺]_i signals for the regulation of long-term potentiation through Ca2+-dependent D-serine release (Henneberger and others 2010). However, in this latter case the source of Ca²⁺ remains to be identified.

Areas for Future Exploration

Our findings on astrocyte TRPA1 channels and their impact on inhibitory synapses were made possible because of the ability of Lck-GCaMP3 to reveal novel spotty Ca²⁺ signals. These findings now lead to three classes of question.

The first class of question centers on the use of GECIs to study astrocyte [Ca²⁺]_i signals within intact preparations and in vivo. Thus, it will be important to extend our approaches with cytosolic and membrane targeted GECIs with in vivo viral expression methods as well as by using transgenic and knock-in expression (Zariwala and others 2012). Our published data strongly suggest these approaches will reveal a new vista of [Ca²⁺]_i signaling in astrocytes that has been missed with conventional bulk loading methods. Specifically, we predict the existence of large numbers of [Ca²⁺]_i signals in branches and branchlets, but it is not clear if any of the available methods will reveal [Ca²⁺]_i signals within the extremely fine astrocyte leaflets that contact synapses (Box 1). Nonetheless, GECIs may provide the metric needed to explore astrocyte functions within intact brain circuits with precision and without the unavoidable problems of organic Ca²⁺ indicator dyes. Additionally, the Lck tag appears to be a wonderful way to recruit fluorescent proteins and sensors to astrocyte plasma membranes and reveal their complex morphology (Benediktsson and others 2005; Shigetomi and others 2010b). Perhaps this approach could be exploited to specifically study astrocyte structural plasticity in the context of physiology, disease, and injury. Structural remodeling of astrocytes is an exciting area that could be fruitfully explored with the Lck tag (Sun and Jakobs 2011).

Although GECIs appear ideal to explore astrocytes, there are limitations. For example, one needs to develop methods to express them within specified astrocytes without changing the physiology of the cells. One needs to ensure that expression of the GECI does not alter Ca²⁺ signaling or interfere with other processes. Finally, one needs to ensure expression of the GECI is reliable and stable over the time course of the experiment at hand. It is likely these requirements can be met by the use of adeno associated viruses (Xie and others 2010) and knock-in mice using Cre-LoxP technology. Floxed STOP mice expressing cytosolic GCaMP3 from the Rosa26 locus are already available (Zariwala and others 2012), and it is likely other mice will soon be made, allowing our approaches with GECIs in vitro to be extended in vivo. Given the strict demands of in vivo imaging discussed above, it is quite possible that further improvements in GECI performance will be required to detect the full complement of in vivo astrocyte [Ca²⁺]_i responses. Just as Lck-GCaMP3 and Lck-GCaMP5G revealed complexities of spotty Ca²⁺ signals not seen with Lck-GCaMP2, next-generation GECIs may uncover additional intricacies or even new classes of Ca²⁺ signals. It is possible that a next-generation GECI, expressed in the cytoplasm, could reveal similar, or improved, signals relative to the current Lck-targeted versions, which would improve compatibility with existing transgenic mice and reagents. Last, further classes of genetically encoded sensors, such as for extracellular glutamate or GABA, combined with GECIs in other color channels (i.e., red), will open new doors to investigate the underlying physiology of astrocytes and neuron-astrocyte two-way communication.

The second class of question centers on the discovery of TRPA1 channels in astrocytes. Specifically, what other functions do astrocyte TRPA1 channels serve? Do they contribute to synaptic plasticity, neurovascular coupling, and/or metabolic support for neurons? Is there an endogenous ligand for TRPA1 channels in astrocytes? Our findings to date suggest that TRPA1 channels have a high open probability at rest and can give rise to spotty Ca²⁺ signals without the need to invoke the existence of an endogenous ligand secreted by, or contained within, astrocytes (Shigetomi and others 2011). However, because TRPA1 channels are activated by a whole host of molecular species, these data do not rule out the possibility that an appropriate ligand may be released under some circumstances to open astrocyte TRPA1 channels, during ischemia, inflammation, and disease, for example. Reliable antibodies against TRPA1 channels are also needed to start systematic anatomical and electron microscopy studies. Additionally, genetic approaches such as cell-specific and inducible deletion of TRPA1 channels may allow us to investigate the roles of these channels in the function of microcircuits and behavior without having to rely on pharmacological approaches. These endeavors would benefit from technical refinements that are desperately needed, such as improved and specific Cre reporter mice that target distinct astrocyte subpopulations.

The third class of question centers on astrocyte GAT-3. Specifically, what are the molecular scale mechanisms that determine why Ca²⁺ entry through TRPA1 regulates functional expression of GAT-3 on the astrocyte surface? What is the cellular substructure that places TRPA1 and GAT-3 within a working distance of ~250 nm? Why does blocking GAT-3 function largely affect mIPSCs onto interneurons and not those onto pyramidal neurons? Do the slice results on mIPSC modulation extend to true, in vivo effects on inhibitory modulation? We hypothesize the existence of closer functional and/or structural interactions of astrocytes with interneurons than with pyramidal neurons in the hippocampal CA1 region: it will be important to test this hypothesis with GAT-3 reporter mice. Because GABA is intimately involved in regulating synaptic inhibition within microcircuits, it will also be important to evaluate if astrocyte TRPA1 or GAT-3 contribute to neuronal hyperexcitability such as during epileptiform activity.

Another general area that could be usefully pursued concerns definition: we need to agree on the magnitude and kinetics of signals that constitute bona fide [Ca²⁺]_i signals, so that we can agree on what each of us in the field is studying. In this respect, the recently reported focal signals appear to be quite small (Di Castro and others 2011) and one wonders if they have previously been missed as noise. A better and agreed definition of what a [Ca²⁺]_i signal is would mitigate such ambiguities.

Perhaps the most exciting class of question that could now be addressed concerns astrocyte diversity. Specifically, diversity of [Ca²⁺]_i signals within single astrocytes and diversity of astrocyte functions within different parts of the brain. It seems quite implausible to us that some of the most populous cells in the brain are a single homogenous morass (i.e., glue). The use of cytosolic and membrane targeted GECIs offers unique opportunities to study astrocyte diversity in genetically specified cell populations and thus begin to explore the fine details and richness of astrocyte biophysics and biology in the brain.

Footnotes

Acknowledgements

Thanks to all members of the lab for comments and discussions. Thanks to Janet Iwasa for help with Figure 4 ().

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The work in the lab was supported by the Whitehall Foundation, the NIH (NS071292, NS060677), the CHDI Foundation, and a Stein/Oppenheimer Endowment Award (BSK).

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Genetically Encoded Calcium Indicators and Astrocyte Calcium Microdomains

Abstract

Keywords

Why We Need to Study Astrocyte Intracellular Ca2+ Signals ([Ca2+]i)

The Need for Improved Methods to Study Diverse Astrocyte Ca2+ Signals

Box 1. On Astrocyte Morphology Nomenclature: Branches, Branchlets, and Leaflets

Genetically Encoded Calcium Indicators and Fluorescence Imaging

GCaMPs and the Lck Tag: Discovery of Spotty [Ca2+]i Signals

Diversity of Ca2+ Signals in Astrocytes

TRPA1 Channels Contribute to Basal Calcium Levels in Astrocytes

TRPA1 Channels in Astrocytes Indirectly Modulate Inhibitory Synapses

Areas for Future Exploration

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Funding

References

Why We Need to Study Astrocyte Intracellular Ca²⁺ Signals ([Ca²⁺]_i)

The Need for Improved Methods to Study Diverse Astrocyte Ca²⁺ Signals

GCaMPs and the Lck Tag: Discovery of Spotty [Ca²⁺]_i Signals

Diversity of Ca²⁺ Signals in Astrocytes