Gray matter protoplasmic astrocytes extend very thin processes and establish close contacts with synapses. It has been suggested that the release of neuroactive gliotransmitters at the tripartite synapse contributes to information processing. However, the concept of calcium (Ca 2+)-dependent gliotransmitter release from astrocytes, and the release mechanisms are being debated. Studying astrocytes in their natural environment is challenging because: (i) astrocytes are electrically silent; (ii) astrocytes and neurons express an overlapping repertoire of transmembrane receptors; (iii) the size of astrocyte processes in contact with synapses are below the resolution of confocal and two-photon microscopes (iv) bulk-loading techniques using fluorescent Ca 2+ indicators lack cellular specificity. In this review, we will discuss some limitations of conventional methodologies and highlight the interest of novel tools and approaches for studying gliotransmission. Genetically encoded Ca 2+ indicators (GECIs), light-gated channels, and exogenous receptors are being developed to selectively read out and stimulate astrocyte activity. Our review discusses emerging perspectives on: (i) the complexity of astrocyte Ca 2+ signaling revealed by GECIs; (ii) new pharmacogenetic and optogenetic approaches to activate specific Ca 2+ signaling pathways in astrocytes; (iii) classical and new techniques to monitor vesicle fusion in cultured astrocytes; (iv) possible strategies to express specifically reporter genes in astrocytes.
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Genetically encoded Ca 2+ indicators Protoplasmic astrocytes are electrically silent. However, they may be considered as excitable cells in the sense that they show Ca 2+ signals, both spontaneously and in response to neuronal activity. In spite of the evidence suggesting that Ca 2+ signals are necessary and sufficient to induce gliotransmitter release, many questions remain, concerning both the role and sources of Ca 2+ signals in astrocytes (Agulhon et al.,; Fiacco et al.,; Parpura et al., ). One limiting factor to study Ca 2+ signaling has been methodological. So far, most studies in acute brain slices and in vivo have been based on bulk-loaded membrane-permeable chemical Ca 2+ indicators. There exist many organic Ca 2+ indicators with different spectral properties and affinity for Ca 2+ which can monitor either Ca 2+-related fluorescence changes or, for the ratiometric probes, can be calibrated to provide absolute Ca 2+ concentration reviewed in (Paredes et al., ). The membrane-permeable acetoxymethyl (AM) esters, Fluo4-AM and Oregon Green BAPTA1-AM (OGB1-AM), the most popular dyes used to image Ca 2+ activity in populations of astrocytes, allow imaging the somatic region and the larger proximal processes but leave the very thin distant processes that participate to the tripartite synapse unsampled (Reeves et al., ).
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At the laser powers, dye concentrations and integration times typically used and with the spatial resolution available, fluorescence changes in fine processes are generally not resolved (see below for a detailed discussion). Another limiting factor has been the lack of specificity of these membrane-permeable Ca 2+ indicators, which label both neurons and astrocytes (Garaschuk et al., ) with cell-type preferences, depending on the indicator, the protocol of application, and the age of the animal. Therefore, sulforhodamine 101 (SR101) that is specifically taken up by astrocytes has been generally used as a secondary fluorescent marker for astrocyte identification. The deep-red emission of SR101 can be detected with negligible spectral overlap with GFP or green fluorescein-based membrane-permeable Ca 2+ indicators (Nimmerjahn et al., ). However, SR101 uptake is age-dependent (Kafitz et al., ) and it does not work in all brain regions (Schnell et al., ). Also, at concentrations needed for astrocyte labeling, SR101 leads to increases of neuronal excitability and long-term potentiation (Kang et al.,; Garaschuk, ), and thus might affect functional studies.
As an alternative, transgenic (Tg) mouse lines (Nolte et al.,; Vives et al.,; Heintz,; Zuo et al.,; Regan et al., ) expressing a green/yellow fluorescent protein (GFP/YFP) or Discosoma red protein (DsRed) under astrocyte-specific promoters (GFAP, S100β, GLT-1, ALDH1L1) can be used to identify astrocytes. In this context, the recent genetically encoded Ca 2+ indicators (GECI) provide a new alternative for non-invasive imaging of Ca 2+ activity in vivo and in brain slices reviewed in (Knopfel,; Looger and Griesbeck, ).
Their level of expression can be stable for months in the absence of apparent adverse effect (Zariwala et al., ). GECIs can be targeted to the plasma membrane of astrocytes (Shigetomi et al., ) and their specific expression by astrocytes in vivo is being developed using viral constructs and Tg mouse lines (see below). Finally new GECI variants are being generated having greater signal to noise ratio, different Ca 2+-binding affinities, and different spectral properties (Horikawa et al.,; Zhao et al.,; Ohkura et al.,; Akerboom et al.,; Chen et al., ) which further enlarge their utility for studying the role of astrocytes on synaptic transmission (Tong et al., ). Among the most recent GECIs, several variants of the original GFP-based Ca 2+ sensor GCaMP1 (Nakai et al., ) have been tested in astrocytes: GCaMP2 (Hoogland et al., ), GCaMP3 (Shigetomi et al.,; Tong et al., ), GCaMP5 (Akerboom et al., ), and red GECIs (Akerboom et al., ), as well as Case12 (Souslova et al.,; Gourine et al., ), and yellow Cameleon YC3.60 (Atkin et al., ). In neurons GCaMP5G and GCaMP6 variants have been shown to produce a higher signal-to-noise ratio than GCaMP3 and can detect Ca 2+ changes evoked by single action potential (Akerboom et al., ). GCaMP3, GCaMP5G, and GCaMP6 are all compatible with two-photon excitation at 910-930 nm (Akerboom et al.,; Mutze et al., ).
Recently, Khakh's group (Tian et al.,; Shigetomi et al., ) compared Ca 2+ changes in astrocytes using a membrane-permeable Ca 2+ indicator (Fluo4-AM) with those detected with two GECIs, the cytosolic GCaMP3 and a membrane-targeted Lck-GCaMP3 (Figure ). Ca 2+ signals were recorded with a confocal microscope at the surface of acute hippocampal slices from adult mice. Specific astrocytic targeting of GCaMP3 and Lck-GCaMP3 was obtained using a short version (gfaABC1D) of the human glial fibrillary acidic (hGFAP) promoter.
Unlike Fluo4-AM which diffuses poorly to the thin astrocyte processes (Reeves et al., ), both GCaMP3 and Lck-GCaMP3 reported a wealth of Ca 2+ signals in distant thin astrocytic processes with relatively less activity in the soma and proximal processes. Expression of GCAMP3 in astrocytes and comparison of Ca 2+ signals detected with Fluo-4, cyto-GCAMP3, and Lck-GCAMP3. (A,B) Illustration of the membrane-targeted Lck-GCAMP3 and cytosolic non-targeted Cyto-GCAMP3. (C) Protocol of AAV2/5 injections into a mouse hippocampus.
(D) Confocal images in CA1 stratum radiatum for Lck-GCaMP3 and cyto-GCaMP3. (E) Colocalization between Lck-GCaMP3 and cyto-GCaMP3 with the astrocytic marker GFAP.
(F) Ca 2+ signals imaged with Fluo-4 (black traces), cyto-GCaMP3 (green traces), and Lck-GCaMP3 (red traces). Top, representative images of astrocytes loaded with Fluo-4AM, Lck-GCaMP3 or cyto-GCaMP3. ROIs are shown in each image, and their time-lapse intensities are shown below. Adapted from (Shigetomi et al., ).
GECIs have been introduced only recently compared to chemical Ca 2+ indicators that have been used since the 80s. Therefore, their photophysical properties and impact on intracellular Ca 2+ homeostasis have not yet been characterized to the same extent as their chemical counterparts (Perez Koldenkova and Nagai, ).
For example, the Ca 2+affinity of many GECIs has not been yet determined in the complex intracellular milieu; their binding kinetics (on- and off-rates for Ca 2+ binding), as well as their aggregation and bleaching rates are not well established. Open questions concern their precise mobility, local concentration, Ca 2+ buffer capacity and subcellular localization, which can be engineered by adding genetically encoded targeting sequences, as done with Lck-GCaMP3 (Shigetomi et al., ). However, the capacity of GECIs to specifically detect local Ca 2+ signals in a population of astrocytes together with their low photobleaching and high signal-to-noise ratio seem to outweigh these limitations.
Indeed experiments become feasible that were simply not possible with earlier small-molecule chemical indicators. The new Ca 2+ data with the GECIs provide intriguing clues to explore astrocyte functions but also present new challenges. New tools will be needed to reliably detect and quantify the wealth of rapid asynchronous and local fluorescence changes. The mechanisms and functional significance of these Ca 2+ signals are far from being understood. Given the striking difference between fluorimetric Ca 2+ signals detected with chemical Ca 2+ indicators and GECIs, the relation between neuronal activity and astrocytic Ca 2+ signals needs to be re-investigated under physiological and pathological conditions using acute brain slices, and anesthetized or non-anesthetized mouse preparations.
Activation of astrocytes Following neuronal activity, the activation of astrocytes is mediated by neurotransmitter released from synaptic terminals (Porter and McCarthy,; Wang et al., ). The subsequent release of gliotransmitters from mature protoplasmic astrocytes has been reported to depend upon G q GPCR activation leading to astrocytic type-2 IP 3 receptor (IP 3R2) activation and Ca 2+ release from the endoplasmic reticulum reviewed in (Halassa et al., ). While this pathway has been implicated in gliotransmitter release, the mechanisms and the concept of gliotransmission remains debated (Agulhon et al.,; Fiacco et al.,; Hamilton and Attwell, ) in part because of our inability to selectively activate Ca 2+ signals in astrocytes. The exogenous generation of Ca 2+ signals that mimic those evoked by neuronal stimuli should clarify the interactions between neurons and astrocytes.
Optogenetics Over the last decade, the development of new photoswitchable genetically encoded channels and receptors to activate and inactivate specific neuronal subtypes had a significant impact on Neuroscience. The simultaneous methodological advances in several fields: (i) optics for photoactivation and imaging in situ, (ii) molecular engineering for developing new photoswitchable proteins, (iii) molecular biology for specific targeting of the light sensitive proteins, have been instrumental for the success of optogenetics in elucidating the function of neuronal circuits (Szobota and Isacoff,; Fenno et al.,; Miesenbock, ). The most popular photoswitchable channel to activate neurons is the H314R channelrhodopsin 2 ChR2(H314R), a variant of the wild type ChR2 with reduced desensitization (Nagel et al., ). ChR2 is a cationic channel highly permeable to proton (P + H/P + Na 10 6) but weakly permeable to Ca 2+ (P 2+ Ca/P + Na 0.117) (Nagel et al.,; Lin et al., ). In neurons, its photoactivation triggers Ca 2+ elevations which depend mainly on the secondary activation of voltage-gated Ca 2+ channels (VGCC) (Nagel et al.,; Zhang and Oertner,; Li et al., ). Attempts have been made to photoactivate protoplasmic astrocytes.
In situ experiments suggest that the photoactivation of ChR2-expressing astrocytes can trigger gliotransmitter release (Gradinaru et al.,; Gourine et al.,; Sasaki et al.,; Chen et al., ). In the rat brain stem retrotapezoid nucleus, ChR2-expressing astrocytes responded to long lasting (20–60 s) illumination by slow Ca 2+ rises that lasted for minutes (Gourine et al., ). In the hippocampal CA1 region, blue light pulses induce rapid time-locked Ca 2+ signals in astrocytes (Chen et al., ). However, our own experiments using mouse cortical astrocytes in culture, show that ChR2 activation induces variable and weak Ca 2+ elevations (Li et al., ). Instead we found that the activation of the Ca 2+-permeable light-gated glutamate receptor (LiGluR) reviewed in (Szobota and Isacoff, ), and the Ca 2+-translocating ChR2 (CatCh) (Kleinlogel et al., ) evokes reliable and robust Ca 2+ signals in astrocytes (Figure ). We attributed the low efficacy of ChR2 in astrocytes to its relatively weak Ca 2+ permeability (Nagel et al.,; Lin et al., ), and to the absence of VGCC in protoplasmic astrocytes (Carmignoto et al.,; Parpura and Verkhratsky, ). Interestingly, LiGluR can be rapidly switched ON and OFF to mimic endogenous Ca 2+ signals recorded with the GCaMP3 (Shigetomi et al., ).
Finally, LiGluR activation induces a large Ca 2+ influx that is further shaped by internal stores, while CatCh activation generates a Ca 2+ influx insensitive to internal Ca 2+ store depletion, indicating that LiGluR and CatCh are interesting tools to activate differentially selective Ca 2+ signaling pathways and to study their downstream effects. Ca 2+ signals recorded in mouse cortical astrocytes in culture using LiGluR, ChR2(H134R) and CatCh photoactivation. (A) Light-gated Ca 2+ rises in an astrocyte expressing LiGluR-mRFP. Ca 2+ rises were imaged with dual-color TIRFM and repetitively switched on and off by 385-nm (violet arrows, 0.3 mW/mm 2, 50 ms) and 488-nm (blue arrows, 39.1 mW/mm 2, 200 ms) light pulses, respectively.
(B) LiGluR(GFP)-gated astrocytic Ca 2+ elevations monitored with the red-fluorescent Ca 2+ dye Xrhod-1. (C) In astrocytes expressing ChR2(H134R), short photoactivation (458-nm, 27.3 mW/mm 2, 500 ms) of ChR2 failed to evoke near-membrane Ca 2+ elevation (top).
Longer light pulses (458-nm, 1 s) evoked variable Ca 2+ signals (bottom). (D) Comparison of the percentage of astrocytes showing light-gated Ca 2+ rises, and of the amplitude of Ca 2+ responses in LiGluR- and ChR2-expressing astrocytes.
LiGluR evokes more reliable and reproducible Ca 2+ rises in astrocytes. (E) CatCh-evoked astrocyte Ca 2+ elevations following blue light photoactivation (1 s, 458-nm). (F) CatCh-induced Ca 2+ signaling was abolished in the absence of extracellular Ca 2+, but unaffected when ER Ca 2+ store is perturbed by thapsigargin (TG). Adapted from (Li et al., ). Astrocytes express a rich repertoire of metabotropic G q, G i/o, and G s GPCRs (Porter and McCarthy, ). New light-gated proteins that mimic these GPCR-mediated pathways have been developed (Schroder-Lang et al.,; Airan et al.,; Ryu et al.,; Gutierrez et al.,; Stierl et al.,; Levitz et al., ), but they have not yet been tested on astrocytes.
Since astrocytes express store-operated Ca 2+ channels Orai1 (Akita and Okada,; Linde et al.,; Moreno et al., ), it should also be of interest to activate them with the new photosensitive synthetic protein LOVS1K that reversibly translocates to Orai1 channels and generates either local Ca 2+ signals at the plasma membrane or global Ca 2+ signals upon repeated photoactivation (Pham et al., ). Optical methods to photoactivate astrocytes While imaging morphological dynamics of astrocytic fine processes may not yet be possible, stimulating astrocytes locally with light is more promising because the astrocyte-specific expression of light-sensitive Ca 2+-permeable ion channels circumvents the optical resolution problem, and therefore even one-photon whole-field illumination is sufficient to stimulate specifically the astrocytes. A more specific photoactivation of a subset of cells, and the local subcellular stimulation of a single astrocyte can be achieved using spatial light modulators reviewed in (Maurer et al., ) to shape the light (Shoham,; Vaziri and Emiliani,; Papagiakoumou, ). One-photon digital holography allows photoactivation within precisely shaped regions of interest at or near the tissue surface. Combining digital holography and two-photon excitation with temporal focusing to modulate the temporal width of the pulsed laser, several groups reported shaped two-photon excitation deep inside scattering tissue (Andrasfalvy et al.,; Papagiakoumou et al., ). The spatial patterns thus generated are robust against scattering and remained confined at depths of 100 μm (Papagiakoumou, ). Combining optogenetics, shaped photoactivation and two-photon imaging for the optical readout of astrocytes (combined with electrophysiology for recording neuronal signals) holds important promises for interrogating interactions between neurons and astrocytes in intact brain tissue.
To probe specific signaling pathways, wave-front light shaping can be combined with uncaging of classical IP 3 and Ca 2+ cages (Ellis-Davies, ), and new endothelin cage (Bourgault et al., ). Monitoring gliotransmitter release Among the mechanisms of gliotransmitter release, Ca 2+-regulated exocytosis of synaptic-like small vesicles has been proposed as a major pathway (Cali et al., ). Total internal reflection fluorescence microscopy (TIRFM) is a powerful technique to monitor single-vesicle behavior and to study the mechanisms of vesicular docking and fusion in cultured cells (Holz and Axelrod, ).
Since cultured astrocytes may differ from their in situ counterparts, the physiological relevance of the findings made in culture with TIRFM need to be validated in situ using other approaches. TIRFM has been used to visualize near membrane single vesicles and monitor single vesicle fusion in cultured astrocytes (Bezzi et al.,; Zhang et al.,; Bowser and Khakh,; Li et al.,; Malarkey and Parpura,; Potokar et al., ). In early experiments (Bezzi et al., ), the fluorescent weak base acridine orange (AO) was used to report exocytosis, and the vesicular glutamate transporter (VGLUT) tagged with the enhanced GFP (EGFP) was overexpressed to identify the AO-positive vesicles. Following DHPG application, rapid millisecond Ca 2+-dependent flashes of AO-labeled vesicles were detected and interpreted as the exocytosis of glutamatergic vesicles (Bezzi et al.,; Domercq et al., ). This interpretation was soon complicated by studies showing that AO metachromasy results in its simultaneous emission of green and red fluorescence, which invalidates the identification of the AO-positive vesicles with EGFP labeling (Nadrigny et al., ). It has also been shown that the flash events of AO-loaded astrocyte vesicles are not solely due to exocytosis but also reflect intracellular vesicle photolysis (Jaiswal et al.,; Li et al., ), due to the action of AO as a photosensitizer.
Styryl pyridinium FM dyes, established markers of vesicular release in neurons (Rizzoli and Betz, ), were also used to label the astrocytic vesicular compartments and report exocytosis. However, FM dyes are handled differently by neurons and astrocytes (Li et al., ) and they label mainly lysosomes (Zhang et al.,; Li et al.,; Liu et al., ). Later, the genetically encoded exocytotic reporter, pHluorin, has emerged as a valuable tool to monitor astrocyte vesicle exocytosis.
As a pH-sensitive GFP mutant, pHluorin fluorescence is quenched in the acidic vesicle lumen and becomes bright upon vesicle fusion when the fluorescent protein is exposed to external neutral pH (Miesenbock et al., ). Since there was evidence that astrocytes release glutamate via Ca 2+-regulated exocytosis (Cali et al., ), pHluorin was targeted to the lumen of putative glutamatergic vesicles in astrocytes by using the fusion protein VGLUT1-pHluorin (Marchaland et al., ). TIRFM imaging of single vesicles in cultured astrocytes labeled with VGLUT1-pHluorin revealed fusion events occurring within hundreds of milliseconds after Ca 2+ rise evoked by either mGluR (Marchaland et al., ) or purinergic P2Y1 receptor activation (Santello et al., ). These results were consistent with those obtained by the same lab using AO-labeled (Bezzi et al.,; Domercq et al., ), and FM-labelled astrocytes (Cali et al., ). Different kinetics have been reported for the exocytosis of the putative glutamatergic vesicles in astrocytes when using another pHluorin-based exocytotic reporter synaptopHluorin (spH), a chimeric construct tagging the luminal side of synaptobrevin 2 (Burrone et al., ). As synaptobrevin 2 appears to colocalize with VGLUT1 on the same vesicles in astrocytes (Montana et al.,; Zhang et al.,; Liu et al., ), expressing spH in astrocytes leads to the labeling of VGLUT1-positive vesicles (Bowser and Khakh,; Liu et al., ).
However, unlike VGLUT1-phluorin that reports fast millisecond kinetics of exocytosis (Cali et al.,; Santello et al., ), spH-labeled vesicles undergo slow exocytosis that is loosely coupled to stimulation, with most events occurring 2 min after P2 receptor-mediated Ca 2+ rise (Malarkey and Parpura, ), and within hundreds of milliseconds following Ca 2+ increase evoked by mechanical stimulation (Liu et al.,; Malarkey and Parpura, ). Genetically encoded reporters of exocytosis set the stage for investigating the mechanisms of astrocyte exocytosis and for addressing several remaining questions. First, the reasons for the variable fusion kinetics of putative VGLUT-positive vesicles remain to be elucidated.
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Second, several studies failed to detect the presence of VGLUT expression in astrocytes (Cahoy et al.,; Juge et al.,; Li et al., ), therefore, new experiments are needed to clarify the molecular identity of the VGLUT-positive vesicles. Recently, a new genetically encoded red pH-sensitive probe, pHTomato, has been introduced to image single vesicle exocytosis (Li and Tsien, ). It should allow monitoring exocytosis, and, simultaneously, activating Ca 2+ signal with optogenetic tools that typically require blue light illumination (Li et al., ). Finally, the new genetically encoded glutamate sensor, iGluSnFR, shows fast kinetics (Marvin et al., ) and is potentially suitable for fast real-time recording of glutamate release from astrocytes. Combining it with TIRFM detection of single vesicle exocytosis would help to clarify the relative contribution of vesicular vs.
Non-vesicular release pathways to glutamate release (Kimelberg et al.,; Li et al.,; Woo et al., ). Fourth, a combination of fast two-photon imaging, local photoactivation and genetically targeted expression of pHluorins in slice and in vivo must validate earlier findings from cell-culture studies. FSc, self complementary, see Gene targeting of viral constructs can also be achieved by the Cre-Lox or tetO-tTA strategies injecting floxed (or flexed) viral constructs in Cre mouse lines, or tetO virus in the tTA mouse lines (Pfrieger and Slezak, ). Several astrocyte-specific Cre- and tTA-expressing mouse lines have been generated (Table ). When a Cre-dependent ChR2-expressing virus was injected in hippocampus (Chen et al., ) of a P14 hGFAP mouse line (Casper and McCarthy, ), selective expression of ChR2 was obtained in the astrocytes.
Tg mouse lines Brain regions studied Specificity References Mlc1-tTA & TetO-ChR2(C128S)-YFP Cerebellum Light-evoked current in the Bergmann glia Sasaki et al.,; Tanaka et al., hGFAP-CreER T2 & Mecp2 +/Stop Whole brain. Yet an important limitation of the viral delivery strategies needs to be taken into account.
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Following intracerebral injection of AAV2/5-gfa104-EGFP construct, a significant dose-dependent reactive gliosis has been observed (Reimsnider et al.,; Klein et al.,; Ortinski et al., ). Since gliosis is associated with changes of several signaling pathways in astrocytes (Hamby et al., ), it will be important to develop alternative approaches to study the role of astrocytes in physiological conditions. Introducing a sequence encoding the VIVIT peptide that interferes with the calcineurin/nuclear factor of activated T-cells signaling pathway and down regulates GFAP overexpression (Furman et al., ), may reduce AAV-induced gliosis. Replacing viral constructs by Tg floxed/tetO mouse lines is a very promising approach to control gliosis. Several floxed (Slezak et al.,; Zariwala et al., ) and tetO (Fiacco et al.,; Agulhon et al., ) mouse lines of interest have been generated.
With a hGFAP-CreER T2 mouse line in which the recombination can be induced in juvenile or adult mice by tamoxifen injections, astrocyte-specific targeting has been obtained in cortex, hippocampal CA1 region, cerebellum, diencephalon and brain stem, with weaker levels of recombination in cortex (Hirrlinger et al.,; Lioy et al., ). In glutamate-aspartate transporter (GLAST)- and connexin 30 (Cx30)-CreER T2 mouse lines, astrocyte-specific recombination occurs in cortex, hippocampal CA1 region, and cerebellum.
But in GLAST-CreER T2 and hGFAP-CreER T2 mouse lines, the recombination is not astrocyte-specific in brain regions including the olfactory bulb and hippocampal dentate gyrus, where neurons are also labeled (Mori et al.,; Slezak et al., ). The floxed/tetO strategy is advantageous since it does not require surgery for viral injections. A Ai38 floxed GCaMP3 reporter mouse line was generated by a knockin strategy to insert GCaMP3 into the ROSA26 locus (Zariwala et al., ). When the Ai38 mouse was crossed with an inducible Wfs1-Tg2-CreER T2 mouse line, a uniform expression of the reporter genes was obtained in cortical excitatory neurons without over expression of GCaMP3 in the nucleus as observed after cortical injection of an AAV-syn-GCaMP3 construct (Figure ). Importantly, it appears that various Cre- and tTA-dependent mouse lines differ in their ability to induce recombination, and also the specificity of recombination can vary with the brain region, and with the age. Therefore, in order to ascertain specificity of astrocytic signal measurement and photoactivation, it will be critical to carefully validate astrocyte-specific expression, for example, by using cell type-specific antibodies and confocal microscopy.