One of the first genes that was removed in astrocytes is the gene encoding glial fibrillary acidic protein (GFAP; Lewis et al., 1984; Reeves et al., 1989; Masood et al., 1993; McCall et al., 1996; Eng et al., 2000). GFAP is a member of the family of intermediate filament structural proteins, which, in the mature nervous system, is found predominantly in protoplasmic and specialized astrocytes of the central nervous system (CNS) as well as in satellite cells, non-myelinating Schwann cells, and enteric glia in the peripheral nervous system (PNS; Jessen et al., 1984; Eng, 1985; Kato et al., 1990). Outside the nervous system, GFAP has also been detected in some rare non-glial cells of the salivary glands (Achstatter et al., 1986; Gustafsson et al., 1989), fibroblasts (Hainfellner et al., 2001), myoepithelial cells (Viale et al., 1991), liver stellate cells (Gard et al., 1985), and lymphocytes (Riol et al., 1997). This protein is one of the key elements of the cytoskeleton that contributes to the morphology and motility of astrocyte processes (Fuchs and Weber, 1994; Pekny and Pekna, 2004; Gomi et al., 2010; Middeldorp and Hol, 2011) and is upregulated in reactive astrocytes (astrogliosis) in essentially any CNS pathology (Eng and Ghirnikar, 1994; Eng et al., 2000; Pekny and Nilsson, 2005; Sofroniew, 2009; Sosunov et al., 2013). During development, GFAP is expressed widely in a number of progenitor cell types giving rise to both neurons and glia. For example, GFAP-expressing radial glia in the ventricular zone (VZ) give rise to mature astrocytes, oligodendrocytes and neurons, as well as guiding subsequent migration of neurons (Gotz et al., 2002; Malatesta et al., 2003; Anthony et al., 2004; Merkle et al., 2004).

The seminal studies reporting the findings obtained with the GFAP KO mouse models were made by targeted deletion of the GFAP gene in ES cells (Pekny et al., 1995; Liedtke et al., 1996; McCall et al., 1996) or by targeted disruption of the GFAP gene by insertion of a LacZ cassette (Gomi et al., 1995). These GFAP KO mouse lines have been used to investigate whether changes in astrocyte processes and their morphological structure can influence brain morphogenesis and function, the physiology of adjacent synapses, and functional recovery after CNS insults. The overall appearance of the GFAP KO mice is indistinguishable from wild-type mice; they develop normally and display no gross alterations in behavior or CNS morphology. This observation suggests at first that GFAP is not essential for normal brain morphogenesis and function. However, closer analyses have indicated the involvement of GFAP in a wide variety of processes. First, these mice display enhanced hippocampal long-term potentiation (LTP) and deficient cerebellar long-term depression (LTD) in acute brain slices from adult mice, suggesting that GFAP intermediate filament protein is important for astrocyte–neuronal interactions and that astrocyte processes play a vital role in modulating synaptic efficacy in the CNS (McCall et al., 1996; Shibuki et al., 1996). In agreement with the cerebellar ex vivo findings, a significant impairment of eye blink conditioning was found in the GFAP KO mice, suggesting that GFAP is required for normal communication between Bergmann glia (specialized astrocytes) and Purkinje cells during induction and maintenance of cerebellar LTD in vivo (Shibuki et al., 1996). Second, morphological and functional alterations in the blood–brain barrier (BBB), disorganization of white matter architecture and vascularization, as well as hydrocephalus were reported in 18–24 month GFAP KO mice, suggesting an involvement of GFAP in the long-term maintenance of normal BBB and CNS myelination (Liedtke et al., 1996). Third, when challenged by diverse brain injuries in vivo, the GFAP KO mice were more vulnerable to: (i) CNS mechanical trauma (Nawashiro et al., 1998; Otani et al., 2006), (ii) cerebral ischemia (Nawashiro et al., 2000; Tanaka et al., 2002), (iii) kainic acid-induced neurotoxicity (Otani et al., 2006), and (iv) autoimmune encephalomyelitis (Liedtke et al., 1998). This was indicated by: (i) increased brain hemorrhage and mortality of mice; (ii) larger cortical infarct volume and profound decrease in cerebral blood flow; (iii) neurodegeneration; and (iv) enhanced clinical course of autoimmune encephalomyelitis and lesions, respectively. Conversely, other investigators have found that suppressing astrocytic GFAP expression in reactive astrocytes increases their basal levels of glial cell derived neurotrophic factor (GDNF), leading to improvement in neuronal survival from metabolic and excitotoxic insults (Hanbury et al., 2003). Beneficial neuroprotective and regenerative effects have also been reported after hippocampal and spinal cord injuries in mice lacking both GFAP and vimentin, another astrocyte intermediate filament protein (Kinouchi et al., 2003; Menet et al., 2003; Wilhelmsson et al., 2004). Collectively, these findings suggest that the GFAP component of the astrocyte cytoskeleton plays an important role in the physiology and pathology of the nervous system. However, because GFAP is expressed in progenitor cells during development giving rise to neurons, oligodendrocytes and astrocytes, these findings have to be viewed carefully. Inducible GFAP KO strategies can be used to circumvent this problem by removing GFAP only from astrocytes after this developmental window has closed (see below).

In light of more recent evidence suggesting that astrogliosis is not a uniform process but rather a multifaceted response with context-dependent reactions (reviewed in Sofroniew, 2009; Sofroniew and Vinters, 2010), the GFAP KO mouse models will continue to be valuable tools in future studies seeking to further unravel the function of GFAP and astrocytes in the variety of human brain challenges or diseases in vivo. These mouse models will assist in understanding the stages when astrocytes are engaged in beneficial or detrimental functions. Furthermore, a number of considerations can be taken into account which may lead to both a re-evaluation of earlier findings using these mice as well as open up new research directions. These include the following: (i) the heterogeneity of astrocyte morphology and physiology (Aley et al., 2006; Emsley and Macklis, 2006; Lee et al., 2006; Matyash and Kettenmann, 2010; Zhang and Barres, 2010; Oberheim et al., 2012; Freeman and Rowitch, 2013; Schreiner et al., 2013); (ii) the variability of GFAP expression levels among this heterogeneous cell population (Bernal and Peterson, 2011; Daniel-Christoph et al., 2013); (iii) the fluctuation of GFAP expression during circadian light–dark cycles, hormonal cycles, developmental, or pathological stages (Hajos, 2008); (iv) the existence of about eight alternatively spliced GFAP isoforms that may execute distinct functions in specific subsets of astrocytes (Middeldorp and Hol, 2011); and (v) the post-translational modification of these different isoforms such as phosphorylation and glycosylation that may influence GFAP assembly into intermediate filaments (Middeldorp and Hol, 2011). Suitable GFAP isoform-specific KO models will be required in future studies to address these issues. One way to investigate the impact of alternative splicing of the GFAP gene in a mouse model would be to selectively delete a single isoform. This can be accomplished by deleting a coding exon from the genome, introducing a stop codon, inactivating the splice sites responsible for generating a specific isoform, or overexpressing a dominant negative version of a certain isoform (Moroy and Heyd, 2007). An alternative way to investigate the impact of alternative GFAP splicing would be to use antisense oligonucleotides to prevent the inclusion of a particular exon in the mature mRNA (Gebski et al., 2003; Dori et al., 2005). Isoform-specific KO mouse models provide a compelling approach to study how alternative splicing of the GFAP gene may contribute to the regulation of pathophysiological CNS processes in vivo.

Another global KO of a gene in astrocytes is the gene encoding the inositol-1,4,5-trisphosphate type 2 receptor (IP₃R2). Two constitutive IP₃R2-deficient mouse models have been obtained through a variation of the gene targeting strategy. In one, the targeting construct was generated by flanking exon 3 of IP₃R2 with two LoxP sites and flanking the neo-cassette by FRT sites (Li et al., 2005). In the other, exon 1 of the gene was fused with a LacZ cassette (Futatsugi et al., 2005). Deletion of the IP₃R2 subtype is a valuable tool in astrocyte research, as it is the only functional IP₃R subtype expressed by astrocytes and the main mechanism by which astrocytes elevate intracellular Ca²⁺ levels (Petravicz et al., 2008; Agulhon et al., 2010; Di Castro et al., 2011; Takata et al., 2011). This mouse line has been critical to address the concept of gliotransmission, which stipulates that neuroactive molecules including neurotransmitters (called gliotransmitters) are released from mature passive astrocytes in a neuronally-induced and Ca²⁺-dependent manner downstream of G_q protein-coupled receptors (G_q GPCRs) to quickly modulate synaptic transmission and plasticity (illustrated and comprehensively discussed in Agulhon et al., 2008, 2012; Hamilton and Attwell, 2010). Upon G_q GPCR activation, IP₃ is produced intracellularly, leading to astrocytic IP₃R2 activation and Ca²⁺ release from the endoplasmic reticulum. Ex vivo and in vivo studies demonstrate that Ca²⁺ transients in mature passive astrocytes are driven by metabotropic G_q GPCRs, which can be activated by the spillover of neurotransmitters released from presynaptic terminals (Porter and McCarthy, 1996; Wang et al., 2006). Therefore, astrocytic G_q GPCRs are considered to be the physiological link between neuronal activity and detectable Ca²⁺ increases in mature astrocytes. However, it is important to keep in mind that future studies using improved imaging methods may reveal alternate sources of activity-driven Ca²⁺ transients in astrocytes. For example, new membrane-tethered genetically encoded Ca²⁺ indicators have allowed for detection of previously unreported constitutive Ca²⁺ transients (Shigetomi et al., 2013b; Tong et al., 2013).

The use of the IP₃R2 KO mouse model was done to selectively obliterate the endogenous G_q GPCR/IP₃R2-mediated Ca²⁺ elevations. The initial studies using this mouse model reported that removing astrocytic G_q GPCR/IP₃R2-mediated Ca²⁺ fluxes did not affect basal and evoked excitatory synaptic transmission (EPSCs), or short- and long-term plasticity (NMDA receptor-dependent LTP) in the hippocampus ex vivo (Fiacco et al., 2007; Petravicz et al., 2008; Agulhon et al., 2010). The implications of these findings have already been well documented and discussed (Agulhon et al., 2008, 2010, 2012; Petravicz et al., 2008; Fiacco et al., 2009a; Hamilton and Attwell, 2010; Kirchhoff, 2010; Nedergaard and Verkhratsky, 2012). Recently, an ex vivo study replicated some of the above-described data by showing that hippocampal NMDA receptor-mediated LTP is normal in IP₃R2 KO mice, supporting the initial findings that the IP₃R2-mediated internal Ca²⁺ store pathway is not involved in activity-evoked gliotransmitter release at the hippocampal CA1–CA3 synapses (Shigetomi et al., 2013b). Moreover, the authors of this study discovered a novel mechanism based on transient receptor potential A1 (TRPA1)-mediated transmembrane Ca²⁺ fluxes, through which astrocytes can modulate LTP. They found that pharmacological blockade or genetic deletion of a recently described TRPA1 channel in astrocytes alters free basal Ca²⁺ levels, leading to a decrease in Ca²⁺-dependent constitutive/homeostatic release of D-serine, and thus LTP reduction – in support of a previous study (Henneberger et al., 2010). Such Ca²⁺ rises are not the type of neuronally-induced astrocyte Ca²⁺ elevations suggested to drive fast release of gliotransmitters by mature astrocytes. Overall these findings emphasize how different astrocyte Ca²⁺ sources (i.e., activity-evoked G_q GPCR/IP₃R2-dependent Ca²⁺ elevations vs. constitutive TRPA1-dependent basal Ca²⁺ dynamics) have distinct effects on LTP (Shigetomi et al., 2013b), and potentially other physiological effects previously attributed to IP₃R2-driven astrocyte Ca²⁺ elevations.

Recent studies using IP₃R2 KO mice in vivo reported that hippocampal muscarinic or cortical NMDA receptor-mediated LTP is diminished or abolished, suggesting a role for IP₃R2-driven Ca²⁺ elevations in synaptic plasticity (Takata et al., 2011; Navarrete et al., 2012). Two exciting new reports are providing a potential explanation for the differences observed between the early and later studies using the IP₃R2 KO. By using a broad range of G_qGPCR antagonists, Wang and coauthors excluded the occurrence of gliotransmission both ex vivo and in vivo (Wang et al., 2012a,b), consistent with the prior ex vivo studies using the IP₃R2 KO mice. Rather, they identified a mechanism by which astrocytic G_q GPCR/IP₃R2-mediated Ca²⁺ elevations stimulate the Na⁺, K⁺-ATPase, leading to a transient K⁺ uptake by astrocytes, a decrease in the extracellular K⁺ concentration, and a subsequent modulation of excitatory postsynaptic currents. This modulation of synaptic transmission was not observed in the IP₃R2 KO mice, implying that astrocyte G_q GPCR/IP₃R2/Ca²⁺-induced decrease of extracellular K⁺ concentration endows astrocytes with a simple and powerful mechanism for rapid modulation of neuronal activity (Wang et al., 2012a,b). Modulation of K⁺ uptake by astrocytic G_q GPCRs was also observed ex vivo (Devaraju et al., 2013; Wang et al., 2013). Rapid modification of K⁺ uptake provides a mechanism which may be responsible for effects on neuronal activity that hitherto have been ascribed to gliotransmission. Nevertheless, synaptic properties are variable within the nervous system, and astrocytes represent a genetically and functionally heterogeneous group of cells (Zhang and Barres, 2010; Oberheim et al., 2012); these cells are likely to exhibit different functions in different areas of the CNS or even within the same area. Therefore, the different effects of knocking out IP₃R2 on neuronal activity support the need to consider heterogeneity of astrocytes between different brain regions when comparing data across studies. Further investigation is necessary to determine the mechanisms involved in activity-induced astrocytic G_q GPCR/IP₃R2/Ca²⁺-mediated modulation of neuronal excitability and LTP in vivo.

In our view, a fundamental and still open question remains to be answered in order to address this issue thoroughly: do neurons express IP₃R2? If so, does this neuronal receptor subtype play a role in specific types of synaptic transmission and plasticity? Fully deleting IP₃R2 abolishes spontaneous and activity- or agonist-dependent G_q GPCR/IP₃R2-mediated Ca²⁺ increases in astrocytes but leaves intact neuronal Ca²⁺ signaling (Petravicz et al., 2008; Di Castro et al., 2011; Takata et al., 2011; Chen et al., 2012; Navarrete et al., 2012; Wang et al., 2012a,b). However, caution should be exercised in the interpretation of the positive physiological data using this mouse model. Indeed, past immunohistochemical studies aimed at identifying the expression of IP₃R2 in neurons were inconclusive (Sharp et al., 1999; Holtzclaw et al., 2002; Hertle and Yeckel, 2007), and most recent antibodies against this IP₃R subtype are of poor quality (Takata et al., 2011; Chen et al., 2012). Additionally, functionally testing whether neurons display IP₃R2-mediated Ca²⁺ signaling would require the use of double KOs for the two other IP₃R subtypes (IP₃R1 and IP₃R3), which is complicated because most of the IP₃R1-deficient mice die in utero (Matsumoto et al., 1996). We are then left with the possibility that some neurons, which also have heterogeneous subtypes throughout the brain, may express IP₃R2 in addition to IP₃R1 and/or IP₃R3. Such a hypothesis, if validated, could help address some of the discrepancies between positive and negative findings using the IP₃R2 KO mice. The use of inducible conditional IP₃R2 KO mice to selectively knock out IP₃R2 in neurons vs. astrocytes in adult mice appears to be the next obvious step to help differentiate between neuronal vs. astrocytic G_q GPCR effects on neuronal excitability and LTP in vivo. Finally, the currently available and future inducible IP₃R2 KO mouse models may help provide insight into brain pathology; recent studies suggest that abnormal (increased) astrocytic Ca²⁺ dynamics are linked to the symptoms of Alzheimer’s disease (Takano et al., 2007; Kuchibhotla et al., 2009; Grolla et al., 2013), suggesting that controlling astrocytic Ca²⁺ homeostasis might be a potential form of therapy for neurodegenerative disease.

Multiple inducible cKO mice have been recently developed to study astrocytic function in vivo. One example is a tamoxifen-inducible cKO mouse specifically lacking cannabinoid type-1 receptor (CB₁R) expression in astrocytes (Han et al., 2012). A mouse line carrying a LoxP-flanked CB₁R (Marsicano et al., 2003) was crossed with a line expressing CreER^T2 under the control of the human GFAP promoter (Hirrlinger et al., 2006). The resulting new line of mice (GFAP-CB₁R-KO) has been used to investigate the mechanisms underlying impairment of working memory, which is one of the most important deleterious effects of marijuana intoxication in humans (Ranganathan and D’Souza, 2006) and animals (Lichtman and Martin, 1996; Wise et al., 2009). Understanding the side-effects associated with the use of marijuana has important clinical implications because the derivatives of marijuana or synthetic cannabinoids represent promising therapeutic molecules for several human conditions, including pain, nausea, seizures, ischemia, cerebral trauma, and tumors (Lemberger, 1980; Carlini, 2004; Hall et al., 2005). The endocannabinoid system has recently emerged as an important neuromodulatory system, and the CB₁R (GPCR predominantly coupling to G_i proteins) is highly abundant in the CNS (Herkenham et al., 1990; Matsuda et al., 1990). In particular, the CB₁R is expressed in glutamatergic and GABAergic neurons (Herkenham et al., 1990; Kawamura et al., 2006) as well as in astrocytes (Navarrete and Araque, 2008; Han et al., 2012) of the hippocampal CA1 area, an area that contributes to spatial working memory (SWM). Because past studies have established that CB₁Rs mediate retrograde inhibition of neurotransmitter release, control neuronal excitability, and regulate short- and long-term plasticity (Di Marzo et al., 1994; Kreitzer and Regehr, 2001; Alger, 2002; Wilson and Nicoll, 2002; Freund et al., 2003; Chevaleyre et al., 2006; Maldonado et al., 2006; Heifets and Castillo, 2009; Kano et al., 2009), research on the function of CB₁R signaling in pain (Kato et al., 2012), aversive memory (Marsicano et al., 2002), epilepsy (Monory et al., 2006), food intake (Bellocchio et al., 2010), analgesia (Zimmer et al., 1999), or development (Jin et al., 2004) has focused mainly on neuronal CB₁Rs using neuronal-specific CB₁R KO mouse models. However, Han et al. (2012) report an unappreciated, yet major, role of astrocytic CB₁Rs in SWM. Their results show that acute cannabinoid exposure in vivo elicits a previously unreported form of LTD at CA3–CA1 hippocampal synapses, which is associated with an impairment of SWM; both effects are abolished in tamoxifen-treated GFAP-CB₁R-KO, but conserved in mice lacking CB₁R in glutamatergic or GABAergic neurons (Han et al., 2012). These findings strongly suggest that astrocytic CB₁R is a primary mediator of cannabinoid-induced LTD. Based on further in vivo pharmacological studies, the authors speculated that their findings are consistent with the possibility that acute exogenous cannabinoid exposure leads to glutamate release from astrocytes, which in turn could activate extrasynaptic NR2B-containing NMDA receptors to trigger AMPA receptor internalization at CA3–CA1 synapses. These events could eventually induce cannabinoid-mediated LTD at these synapses, altering hippocampal SWM. However, several points should to be kept in mind when considering this interpretation of the data, which might imply a substantially more complex mechanism at the CA1–CA3 synapses: (i) the apparent mechanistic discrepancy between this in vivo cannabinoid-induced LTD and previous ex vivo “gliotransmission” studies suggesting that astrocytic CB₁R-mediated Ca²⁺ increases trigger the release of glutamate to activate pre-synaptic mGluRs that leads to glutamate presynaptic release and subsequent potentiation of postsynaptic NMDA receptor-mediated currents (Navarrete and Araque, 2008, 2010); (ii) the current debate as to whether astrocytes actually release glutamate in a GPCR/Ca²⁺-dependent manner in vivo (Wang et al., 2012a compared to Navarrete et al., 2012); (iii) the emerging evidence that astrocyte GPCR/Ca²⁺-dependent release of glutamate occurs in the early steps of inflammatory processes rather than during normal physiology (Agulhon et al., 2012; Pascual et al., 2012); (iv) the recent findings suggesting that astrocytic GPCR/Ca²⁺ triggers an increase of K⁺ uptake (Wang et al., 2012a,b; Devaraju et al., 2013); and (v) a past study reporting that LTD is modulated by astrocytic K⁺ uptake (Janigro et al., 1997). Further investigation should help determine whether cannabinoid-induced LTD is due to an increase of extracellular glutamate, a decrease of extracellular K⁺, a combination of both, or some other mechanism.

Astrocytes become reactive in nearly all brain pathologies, and play important roles in neuroinflammation, a common feature of the aging brain and most neurological disorders and neurodegenerative diseases (Sofroniew, 2009; Sofroniew and Vinters, 2010). Changes in reactive astrocytes include upregulation of GFAP expression (Pekny and Nilsson, 2005), secretion of inflammatory mediators from astrocytes (Lucas et al., 2006; Peng et al., 2006; Farina et al., 2007; Brambilla et al., 2009; Steele and Robinson, 2012), and altered expression of astrocytic GPCRs (Aronica et al., 2003; Hamby et al., 2012). One such GPCR is the dopamine D2 receptor (DRD2) that couples to G_i (Missale et al., 1998) and is expressed not only in neurons, but also in astrocytes (Bal et al., 1994; Khan et al., 2001; Luo et al., 2009). Downregulation of DRD2 expression has been reported in the brain of the elderly (Kaasinen et al., 2000), suggesting that astrocytic DRD2 signaling may be involved in neuroinflammation that occurs in the CNS during aging and disease. A recent study has investigated this question using both astrocyte-specific cKO and tamoxifen-inducible cKO mouse models (Shao et al., 2013). Several complementary methodological approaches have been used, including immunohistochemistry, biochemistry, and molecular biology to show that DRD2-deficient astrocytes show robust GFAP upregulation in the substantia nigra of aged mice. Moreover, DRD2-deficient astrocytes were found to produce more proinflammatory mediators than their wild-type counterparts, suggesting that astrocytic DRD2 is a key negative regulator of neuroinflammation. To identify the downstream effectors of DRD2 that might be involved in the regulation of inflammatory mediator production, microarray analysis was carried out and showed a pronounced decrease of the small heat-shock protein αB-crystallin (CRYAB) in DRD2-deficient astrocytes, a protein known to display anti-inflammatory and neuroprotective activities (Ousman et al., 2007; Bhat and Steinman, 2009). These findings suggest that activation of astrocytic DRD2 controls CRYAB expression to suppress inflammatory responses in astrocytes and thus contribute to the maintenance of the immune state balance under normal conditions (Shao et al., 2013). The implications of these findings are profound in age-related diseases, where a decrease of DRD2 signaling in astrocytes may lead to an abnormal increase of proinflammatory mediator release, and underlie the progression of cognitive and motor function impairments. Therefore, the astrocyte DRD2 signaling pathway could be a potential target for therapy of several neurological, neuroinflammatory, and neurodegenerative disorders, in which production of inflammatory mediators is a common element (Lucas et al., 2006).

It is interesting, however, to note that activation of another G_i GPCR, called CXCR4 in cultured cortical astrocytes has previously been reported to increase release of the proinflammatory cytokine tumor necrosis factor α (TNFα; Bezzi et al., 2001), and not to negatively control the release of proinflammatory molecules as reported by Shao et al. (2013). Several obvious possibilities may explain these differences: (i) astrocytic G_i signaling triggers different cellular mechanisms depending on whether the studies are performed in vivo vs. ex vivo cultures; (ii) astrocytic G_i signaling leads to distinct responses depending on the area of the brain (e.g., substantia nigra vs. cortex); or (iii) astrocytic G_i GPCRs are involved in different functions depending on the developmental stage (mature vs. immature astrocytes). Additionally, another possibility that has not been addressed thoroughly in the field when investigating the role of astrocyte signaling in general is that different astrocytic G_i GPCRs may activate distinct signaling pathways even though they are known to couple to G_i proteins. In other words, depending on the endogenous or synthetic agonist used to active a specific G_i GPCR, this receptor may show a different bias toward other G proteins over G_i or different functional selectivity for β-arrestin recruitment over G_i activation (Allen et al., 2011; Zidar, 2011; Audet et al., 2012; Blattermann et al., 2012; Hiller et al., 2013). The depth of the complexity of GPCR signaling is now becoming familiar territory to receptor biologists, yet the application of this knowledge to the astrocyte field remains extremely limited. Therefore, findings relative to astrocytic GPCR signaling (coupling to G_q, G_i, or G_s proteins) in physiology and pathology likely reflect the complex interactions of multiple signaling pathways and provides an explanation for seemingly contradictory observations. Examples of this have already been observed relative to astrocyte Ca²⁺ signaling which is traditionally ascribed to G_q GPCR signaling pathways, yet astrocytic G_i-coupled GABA_B and CB₁Rs also have been reported to evoke Ca²⁺ elevations in astrocytes (Serrano et al., 2006; Navarrete and Araque, 2008). Future studies using biased ligands in combination with astrocyte-specific cKOs of specific G proteins or β-arrestins is one approach to elucidate the role of astrocyte signaling molecules in brain function and dysfunction.

One important limitation in addressing the role of astrocytic GPCR signaling in neurophysiology has been the inability to pharmacologically activate astrocytic GPCRs in a cell type-specific manner. Indeed, astrocytes in vivo express members of most of the different families of GPCRs linked to the diverse array of intracellular signaling cascades (Porter and McCarthy, 1997) that are also known to be expressed by neurons. Therefore, the use of pharmacological approaches consisting of agonist application ex vivo or in vivo does not allow cell-specific GPCR activation. Therefore, interpretation of the findings is made difficult by direct activation of neuronal (but also other cell type) receptors by the applied agonist in addition to the intended astrocytic targets. As a consequence, it has been difficult to determine the effect of selectively stimulating astrocytic G_q GPCR-mediated Ca²⁺ signaling cascades on physiological processes such as synaptic transmission and plasticity. This complication has led investigators to consider previous reports of gliotransmission with caution.

Thus, advances in this field depend upon the development of novel tools to better address the physiological relevance of astrocytic G_q GPCR Ca²⁺ signaling. In order to overcome some of the limitations associated with traditional pharmacological approaches, two novel transgenic mouse models were developed (Fiacco et al., 2007; Agulhon et al., 2013). In the first model, a novel G_q GPCR is expressed selectively in astrocytes that is not expressed by other cell types in the brain, is not activated by endogenous ligands released in brain, and whose ligand, the peptide FMRF, does not activate endogenous brain G_q GPCRs (Fiacco et al., 2007). The novel receptor, the so-called Mas-related gene A1 (MrgA1), is a member of a family of GPCRs normally expressed in specific subsets of nociceptive sensory neurons in the spinal cord (Dong et al., 2001), but is not found in the brain. The MrgA1 receptor is targeted to astrocytes using the inducible Tet-Off system (Figure 2B). In one transgenic mouse line the green fluorescent protein (GFP)-tagged MrgA1 receptor is transcribed from the TetO promoter. When crossed with a second transgenic line in which tTA is targeted to astrocytes using the human GFAP promoter, a bigenic line (referred to as the MrgA1 mice) is obtained, in which MgrA1-GFP is selectively expressed in the vast majority of astrocytes in the absence of doxycycline (Fiacco et al., 2007). Several studies using this novel transgenic MrgA1 mouse model have shown that increasing astrocytic MrgA1-mediated Ca²⁺ fluxes does not lead to gliotransmission ex vivo and in vivo, suggesting that astrocytes do not release gliotransmitters in a G_q GPCR/Ca²⁺-dependent manner (Fiacco et al., 2007; Agulhon et al., 2010; Wang et al., 2012a,b). Rather, it has been reported that astrocytic MrgA1R-mediated Ca²⁺ elevations potentiate astrocyte glutamate and K⁺ uptake (Wang et al., 2012a,b, 2013; Devaraju et al., 2013), suggesting that the mechanisms by which agonist-induced astrocyte G_q GPCR activation modulates neuronal activity may be different than previously thought, in agreement with previous and most recent findings (Fiacco et al., 2007; Agulhon et al., 2010; Wang et al., 2013). It is important to bear in mind that agonist-evoked stimulation of G_q GPCRs, including MrgA1Rs, generates Ca²⁺ elevations that may not recapitulate some endogenous Ca²⁺ elevations in astrocytes which often remain confined to fine processes. One strategy in future studies could be to employ a caged version of FMRF to locally stimulate astrocyte Ca²⁺ elevations by activation of a G_q signaling pathway, which may more closely resemble the subset of microdomain astrocytic Ca²⁺ elevations which have been observed in astrocytes in vivo.

More recently, another transgenic mouse line has been developed in order to facilitate investigation of the role of astrocytic G_q GPCR signaling in vivo (Agulhon et al., 2013). One of the limitations of the MrgA1 mice is that the MrgA1 FMRF ligand does not cross the BBB, making in vivo studies more invasive as surgeries would be required in order to infuse FMRF into the brain of freely moving mice. Additionally, endogenous MrgA1 is expressed in sensory nerve terminals, preventing the use of MrgA1 mice to study spinal cord astrocytes (as FMRF may diffuse and activate endogenous MrgA1 in nociceptive sensory neurons). To overcome some of these obstacles, a new GFAP-hM3Dq mouse line was created (Agulhon et al., 2013). The innovation of this mouse line is based on the use of a novel genetically engineered G_q GPCR (called hM3Dq) that does not respond to endogenous ligands, but instead responds to an inert synthetic ligand (clozapine-N-oxide, CNO) that crosses the BBB and activates signaling cascades in a similar fashion as endogenous G_q GPCRs (Armbruster et al., 2007). Such new chemogenetic technology (called designer receptors exclusively activated by a designer drug, or DREADD) has recently led to several important discoveries in neuronal function (e.g., Alexander et al., 2009; Ferguson et al., 2011; Atasoy et al., 2012; Garner et al., 2012), and is now being applied to astrocytes for the first time (Agulhon et al., 2013). This mouse line was made through conventional transgenesis using a construct containing the human GFAP promoter driving the expression of hM3Dq (Figure 2A). Inducible and reversible expression of the transgene is not a constraint when using the DREADD technology, as hM3Dq can be activated postdevelopmentally through intraperitoneal CNO injection. Immunohistochemical screening in adult mice indicates that expression of hM3Dq is restricted to astrocytes within the CNS, and in non-myelinating Schwann cells of sympathetic, sensory, and sciatic nerves, as well as satellite cells in sympathethic, parasympathetic, and sensory ganglia within the PNS. Such expression is expected for a GFAP-driven transgene and allows CNO-induced global stimulation in GFAP⁺ glial cells within the CNS and PNS. This provides a powerful approach, as it can reveal most of the physiological and behavioral phenotypes mediated by GFAP⁺ glial cell G_q GPCR signaling. Acute CNO intraperitoneal injections of GFAP-hM3Dq mice resulted in previously unreported and long-lasting (minutes to hours) modulation of autonomic nervous system (ANS) function, including increased heart rate, blood pressure, and saliva formation, as well as decreased body temperature. Furthermore, changes in activity-related behavior and motor coordination were observed in CNO-treated GFAP-hM3Dq mice. To address whether ANS and activity-related effects were due to Ca²⁺ or other signaling pathways downstream of G_q GPCR activation, the GFAP-hM3Dq mice were crossed with IP₃R2 KO mice in order to generate GFAP-hM3Dq mice in which IP₃R2-dependent Ca²⁺ increases were abolished. Interestingly, CNO-treated GFAP-hM3Dq/IP₃R2-deficient mice exhibited similar autonomic and motor modulation as CNO-treated GFAP-hM3Dq mice. These findings suggest that CNO-induced phenotypes are not dependent on IP₃R2-dependent Ca²⁺ increases. Thus, other (non-Ca²⁺) signaling molecules activated by G_q GPCRs in GFAP⁺ glial cells may be important contributors to the functional effects of CNO. Collectively, these findings open new avenues into investigation of GFAP⁺ glial cell (astrocyte, non-myelinating Schwann cell, satellite cell) function in animal physiology. Further studies employing local CNO infusion into specific regions of the CNS or PNS, and specific blockers of G_q GPCR signaling molecules (e.g., protein kinase C or βγ-dependent activation of signaling cascades), will help dissect out the areas of the nervous system and the signaling pathways that are responsible for each of the CNO-induced phenotypes. Viral transduction of hM3Dq in wild-type mice is also a potential strategy to help determine what areas of the CNS vs. PNS are involved in the effects observed in CNO-treated GFAP-hM3Dq mice. Finally, when specific markers of different subpopulations of CNS astrocytes become available, it will be possible to use new promoters to create transgenic mice expressing hM3Dq in distinct subsets of astrocytes. This will allow determination of whether a specific subset of astrocytes is responsible for all of the phenotypes observed in CNO-treated GFAP-hM3Dq mice, or whether different subsets of astrocytes are responsible for distinct phenotypes. Clearly, there is much to be learned with regard to the role of astrocytes, and more generally of GFAP⁺ glial cells, in complex physiology and behavior. Collectively, the findings of this study point to astrocytes as potential therapeutic targets for some ANS or motor dysfunctions.