Optogenetic experimentation on astrocytes M. Figueiredo1*, S. Lane1*, F. Tang1, B.H. Liu1, J. Hewinson1, N. Marina2, V. Kasymov2, E. A. Souslova3, D.M. Chudakov3, A.V.

Gourine2, A.G. Teschemacher1, S. Kasparov1

1. School of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, BS8 1TD, UK. 2. Neuroscience, Physiology & Pharmacology, University College London, London WC1E 6BT, UK 3. Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Miklukho-Maklaya 16/10, 117997, Moscow, Russia * - These authors made equal contributions to this paper

Correspondence to: S. Kasparov. School of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, BS8 1TD, UK. E-mail: sergey.kasparov@bristol.ac.uk Tel +44 117 3312275

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Abstract We briefly review the current literature where optogenetics has been used to study various aspects of astrocyte physiology in vitro and in vivo. This includes both, genetically engineered Ca2+ sensors and effector proteins such as channelrhodopsin. We demonstrate how the ability to target astrocytes with cell-specific viral vectors to express optogenetic constructs helped to unravel some previously un-suspected roles of these inconspicuous cells.

1. Introduction Optogenetics is an emerging technology combining optical methods and molecular biology, which can be used to both optically monitor various processes in the cells of interest or control their activity by light. This brief review will summarise recent advances achieved in both of these areas of optogenetics and discuss how their implementation may help to better understand how astrocytes communicate with each other and with adjacent neuronal networks. For a long time, glial cells were considered passive elements in the brain, providing structural and metabolic support to neurones. Novel experimental evidence, however, suggests that glial cells are anything but passive; they are now regarded as critical elements that sense and respond to neuronal activity and actively participate in information processing (Araque et al., 1999). Astrocytes are the most abundant type of glial cell in the brain and are closely associated with both blood vessels and neurones. A single astrocyte may contact numerous synapses (Bushong et al., 2002), potentially regulating neuronal activity and synaptic transmission (Haydon & Carmignoto, 2006;Pascual et al., 2005).

Astrocytes lack the ability to generate action potentials and thus do not communicate via propagating electrical signals. Instead they respond to stimulation by intracellular calcium [Ca2+]i transients or waves which is frequently referred to as Ca2+ excitability and which have been recorded in vivo (Bekar et al., 2008;Schummers et al., 2008;Hirase et al., 2004;Shigetomi et al., 2010;Wang et al., 2006;Dombeck et al., 2007), in vitro (Cornell-Bell et al., 1990), and also in human brain slices (Oberheim et al., 2009) using conventional chemical Ca2+ indicators. Therefore Ca2+ signalling in astrocytes is seen as an important clue to their physiological roles. Due to the limitation in current methodology, the measurement of [Ca2+]i in small volume compartments such as astrocytic processes and peri-membrane micro-domains transients has

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been somewhat hampered; instead most studies concentrated on the total cytoplasmatic [Ca2+]i in somatic regions of astrocytes (Grosche et al., 1999;Nett et al., 2002). To resolve peri-membrane Ca2+ microdomains with Fluo-4 or x-Rhod-1, a special technique such as total internal reflection fluorescence (TIRF) was necessary (Marchaland et al., 2008). In contrast to the conventional chemical Ca2+ indicators, genetically encoded Ca2+ sensors can be expressed in astrocytes selectively (Gourine et al., 2010) and targeted to specified cellular compartments, for example plasma membranes, by using well characterised targeting motifs (Shigetomi et al., 2010). Recently published data based on these approaches is presented in the first part of this review.

Optogenetics also offers numerous light-sensitive proteins as tools for selective activation (and possibly de-activation) of astrocytes (Nagel et al., 2005). Such tools can be used to manipulate these cells in the context of heterogeneous brain tissue and shed light on what physiological responses they actually trigger in vitro and in vivo. Astrocytes can affect the functions of neuronal networks in a variety of ways. A full account is beyond the scope of this review, but briefly, the currently discussed hypotheses include: release of glio-transmitters such as ATP, glutamate and D-serine; increased or decreased production of lactate which may be an important metabolic substrate and messenger in astrocyte-neurone communication (Magistretti, 2006); changes in the activity of uptake mechanisms; changes in prostaglandin signalling (Gordon et al., 2007;Gordon et al., 2008). Optogenetic control of astrocytic activity and function is discussed in the second part of this brief review.

Finally, as all successful optogenetic experiments on astrocytes in vivo, published to date have been utilizing cell-specific viral vectors for gene delivery we will briefly describe the currently available options. 2. Optical analysis of astrocytic [Ca2+]i signalling Genetically Encoded Calcium Indicators (GECI) consist of one or two fluorescent protein(s) (FP) and a Ca2+-sensitive domain. In the presence of Ca2+, GECI respond by altering their fluorescence intensity or by a wavelength shift.

2.1 Single GFP-based biosensors The chromophore of GFP is enclosed in a tight -barrel structure which is essential for GFP to be fluorescent and makes access to its internal space particularly difficult (Yang et al., 1996). Yet,

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interruptions at certain positions, circular permutation or even insertion of foreign proteins still may result, with appropriate optimization, in functional constructs. The most common single GFP-based biosensor platform relies on circularly permuted GFP (cpGFP) (Baird et al., 1999), in which GFP is rearranged so that the original N- and C termini are connected with a flexible peptide linker and another peptide bond is interrupted to form new N and C-termini which contain sticky ends the Ca2+ binding motif from Calmodulin (CaM) and its target binding protein, M13 (derived from myosin light chain kinase; Figure 1A). The linker sequences used to fuse M13 and CaM to the cpGFP moiety and the linker in the middle of the molecule are critical for tuning the response properties of cpGFP-based Ca2+ indicators (Leder et al., 2010). An ideal indicator from this family should increase its fluorescence proportionally to Ca2+ concentration (Figure 1B left side). In 2001 Nakai and his colleagues generated GCaMP (Nakai et al., 2001) (Figure 1A). The first version of GCaMP displayed dim fluorescence at resting states, poor folding, and slow maturation at 37C, as well as nonlinear bleaching (Nakai et al., 2001;Lin et al., 2004). Furthermore, in the presence of Ca2+, the GCaMP absorption spectrum changed (Nakai et al., 2001). Subsequent modifications led to generation of GCaMP1.6 (Ohkura et al., 2005) and GCaMP2 (Tallini et al., 2006) demonstrating a 5-fold and 6-fold fractional fluorescence change, respectively, of its predecessor (please note that these values reflect the results of in vitro tests in cell-free systems) . The most recently published addition to that family is GCaMP3, which has a further improved dynamic range (10 fold increase in fluorescence between resting and stimulated state) (Tian et al., 2009). In 2007 another cpGFP-based indicator was introduced (Souslova et al., 2007), named Case12 because of its exceptionally high contrast ratio (12 times that of resting to fully Ca2+ bound state). This sensor so far is the only one successfully used for astrocytic [Ca2+]i imaging not only in vitro (Guo et al., 2010) but in vivo (Gourine et al., 2010). Given that in cultured astrocytes un-targeted GCaMP2 was not very effective (Shigetomi & Khakh, 2009), it could be that the lower Ca2+ affinity of Case12 may be advantageous in vivo. Current cpGFP-based Ca2+ sensors have high dynamic ranges but they also have limitations. First of all, opening of the fluorescent barrel allows entry of protons into the internal cavity and inevitably makes them pH sensitive. For Case12 and GCaMP1.6 the pKa is ~7.2, which is quite close to the physiological range (Souslova et al., 2007). For GCaMP2 and 3, we were unable to find this information, but it could be postulated that they are also pH sensitive. Such pH sensitivity can complicate interpretation of some experiments, as illustrated below (Section 1.3).

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Secondly, photostability of cpGFP may be insufficient for some applications. Case12 undergoes a fully reversible quenching when exposed to high intensity blue light (Souslova et al., 2007) but its fluorescence fully recovers after a few seconds of darkness. Tian and colleagues (Tian et al., 2009) imaged GCaMP3 using a two photon excitation microscope with 10 mW of laser power at the specimen and in this test photostability of GCaMP3 was superior to some other FPs. However, because illumination was not continuous but interspersed by 30 second episodes of darkness, it is difficult to correctly assess the dynamics of its bleaching and/or reversible quenching.

2.2 FRET-based sensors Frster resonance energy transfer (FRET) was, in fact, the first successful approach to generation of Ca2+ biosensors. In 1997 two groups reported constructs where CaM and M13 motifs were used to alter the conformation of a molecule which incorporates a FRET pair of FPs with different excitation and emission characteristics (Miyawaki et al., 1997;Persechini et al., 1997;Romoser et al., 1997)(Figure 1B right and 1C). The disadvantage of CaM-based Ca2+ sensors is that neurones in particular may express other potential substrates with which Ca2+bound CaM may interact, and this applied equally to the cpGFP-based sensors discussed in the previous section. Obviously such interaction will prevent the conformational change required to induce FRET upon [Ca2+]i increase (Palmer & Tsien, 2006). The most recent constructs from this sub-family were reported to detect Ca2+ transients triggered by single action potentials (Wallace et al., 2008;Horikawa et al., 2010a). One elegant way around the problem of un-solicited interactions of CaM with endogenous proteins is to use troponin C as a Ca2+ sensing moiety, because outside of the skeletal muscle it seems to have no protein targets to interact with. This approach has been realised in TN-XXL (Mank et al., 2008)(Figure 1D). FRET-based sensors also work in membrane-targeted fusions (Nguyen et al., 2010). To the best of our knowledge these indicators have not yet been used to monitor astrocytic [Ca2+]i, although they may offer some advantages, for example much better pH stability. 2.3 Monitoring [Ca2+]i signalling in astrocytes using GECI GCaMP2 (Tallini et al., 2006) was used to image [Ca2+]i signals in microdomains at the plasma membrane of cultured astrocytes by fusing it to the subunits of the Na+ pump (Lee et al., 2006). This fusion helped to reveal highly focal Ca2+ signalling in the areas of the membrane adjacent to the junctional endoplasmatic reticulum (Lee et al., 2006). In a recent study, GCaMP2 and

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GCaMP3 were fused to the membrane-targeting domain of the tyrosine kinase Lck which contains palmitoylation and myristoylation domains acting as membrane anchors (Shigetomi et al., 2010). In cultured astrocytes only Lck-targeted sensors revealed localised [Ca2+]i transients in domains as large as 510 M near the plasma membrane (consistent with (Marchaland et al., 2008)). These transients either occurred apparently spontaneously in which case they were insensitive to a blocker of neuronal action potentials, tetrodotoxin (TTX), or could be evoked by activation of co-cultured neurones by current pulses, in which case they were sensitive to TTX and likely mediated by ATP (Shigetomi et al., 2010).

We have generated an adenoviral vector (AVV) which selectively express Case12 in astrocytes (AVV-sGFAP-Case12), using an amplified shortened promoter of the glial fibrillary acidic protein (GFAP) (see section 3). The affinity of Case 12 to Ca2+ is lower (Kd ~1 mkM) than that of GCaMP2 and 3 (~50 nM in (Shigetomi & Khakh, 2009)). This might be advantageous for Ca2+ imaging in astrocytes because we easily detect spontaneous Ca2+ activity in cultured astrocytes with Case12 while Shigestomi et al 2009 failed to reveal any. If this supposition is correct, then the very recently developed nano-Cameleons (Horikawa et al., 2010b) could be best suited for imaging neurones but not astrocytes. When expressed in astrocytes (Figure 2A-C) Case12 sensitivity compares to that of the conventional chemical Ca2+ indicator, Rhod-2AM (Guo et al., 2010;Gourine et al., 2010). Case12 fluorescence faithfully follows the signal derived from a conventional indicator Fura-2 (Figure 2C). At the same time Case12 is pH sensitive (note that the intensity of the signal falls below baseline at the end of the pH stimulus in Figure 2Bi and 2C). Case12 visualises changes in [Ca2+]i not only in somata but also in the finest astrocytic processes (Gourine et al., 2010). In Guo et al., 2010, AVV-sGFAP-Case12 was used to study the effects of one of the neuro-hormones from the angiotensin family, angiotensin 1-7 (Ang 1-7). The application of this viral vector to brainstem slice cultures resulted in selective expression of Case12 in astrocytes, thus defining the source of the optical signal in the imaging experiments. In addition, neurones were labelled with other viral vectors incorporating different promoters. We were able to show that Ang 1-7 activates a fraction of astrocytes without affecting local neurones. One great strength of the viral vector approach is its applicability to different animal models and we were able to compare the effects of Ang 1-7 between normotensive and hypertensive rat strains in vitro (Guo et al., 2010).

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AVV-sGFAP-Case12 was also used in vivo to reveal the chemosensitivity of a specific subset of astrocytes located on the ventral surface (VS) of the medulla oblongata (Gourine et al., 2010). We found that a 0.2 pH unit decrease on the VS of anaesthetised and artificially ventilated rats evoked an immediate increase in [Ca2+]i across the field of VS astrocytes. On the other hand, in cultured slices, we observed that acidification-induced Ca2+ excitation of VS astrocytes was insensitive to TTX and muscimol (Figure 2B), two agents which silence retrotrapezoid nucleus neurones, the only known type of pH-responsive neurones in this area. Hence we were able to pinpoint local astrocytes as the primary chemoreceptors of the VS. Moreover, using [Ca2+]i responses in astrocytes as a means of detecting ATP-mediated signalling, we found that acidification triggered release of ATP. Indeed the ATP-degrading enzyme apyrase almost completely abolished these [Ca2+]i waves (Gourine et al., 2010). Therefore, propagation of pHevoked Ca2+ excitation among ventral medullary astrocytes is largely mediated by ATP acting on a subset of P2Y receptors.

In summary, GECIs have proven to be highly valuable tools for in vitro and in vivo imaging of astrocytic [Ca2+]i and any new members of this family developed to overcome the current limitations hold high promise for future research. 3. Optical control of astrocytic [Ca2+]i and transmitter release 3.1 Channelrhodopsin-2 To optogenetically control their functional activity; light-activated constructs can be targeted to selected cell types. A number of recent reviews summarise origins, features of currently available variants of the channelrhodopsin family and their application to experiments on neurones (see other reviews in this issue). Channelrhodopsin-2 (ChR2) is an algal light-gated cation-selective membrane channel. Its molecule consists of a 7-transmembrane-spanning apoprotein, channelopsin, and retinal which covalently binds to it (Bamann et al., 2008;Wang et al., 2009). After absorption of a photon ChR2 opens rapidly to form a pore permeable to monovalent (Na+, K+, H+) and divalent cations (Ca2+). The high light-induced cation conductance leads to strong and rapid depolarisation of the membrane (Nagel et al., 2003). It is thought that the photoisomerization of all-trans-retinal to 13-cis configuration induces the protein to change its conformation and open the channel (Hegemann, 2008). Various mutants of ChR2 are currently available (see review by J Lin in this volume (Lin, 2010)) including ChR2(H134R)

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(Nagel et al., 2005;Gradinaru et al., 2007). We have selectively expressed ChR2(H134R) in astrocytes under the control of an enhanced GfaABC1D promoter (see section 3). In order to visualise transduced astrocytes without concomitant excitation, we fused ChR2(H134R) to a redshifted fluorescent protein, Katushka1.3 (later re-named as Katushka 2). Katushka1.3 has its emission peak at ~630 nm (Shcherbo et al., 2009) and can be excited by green or yellow laser light, thus avoiding activation of ChR2(H134R) by blue light. Moreover, the spectral properties of Katushka1.3 allow us to use Rhod-2AM, a red-shifted Ca2+ indicator, in combined experiments with ChR2(H134R) (Figure 3A).

In vitro activation of astrocytes was achieved with moderate intensity blue light (470 nm laser diode microscope illumination system from Rapp OptoElectronic, Germany). In order to illuminate the cells and at the same time continue using green (543nm) or yellow (568nm) laser we modified the light path of our confocal imaging system (SP2 Leica confocal upright microscope) and added an additional dichroic mirror (Figure 3B).

3.2 Validation of AVV-sGFAP-ChR2(H134R)-Katushka1.3 To verify the ability of our vector, AVV-sGFAP-ChR2(H134R)-Katushka1.3, to excite astrocytes, several in vitro tests were performed. First, we loaded dissociated cultured astrocytes with Rhod-2AM and monitored fluorescence using a SP2 Leica confocal microscope (Figure 3B). Flashing blue light (470 nm, 20/20 msec duty cycle) was used to activate ChR2(H134R). This caused increases in [Ca2+]i in transduced astrocytes (Figure 3C). It is important to note that in different experiments the speed of [Ca2+]i rises and their dynamics (fast, slow recovery or no recovery during the observation period) were different and this depended on the expression level of ChR2(H134R) as well as the light intensity used for stimulation. In order to determine the source of the [Ca2+]i, astrocytes were superfused with Ca2+-free buffer with added EDTA. This resulted in an almost complete elimination of light-induced [Ca2+]i increases, suggesting its dependence on the influx of Ca2+ from the extracellular space (Figure 3D).

The next step was to determine if the new vector could be used to trigger release of ATP in a more integrated preparation. Organotypic brain slices were prepared, as previously described (Teschemacher et al., 2005b) and transduced with AVV-sGFAP-ChR2(H134R)-Katushka1.3. After 7 days, slices were placed into a tissue chamber, continuously superfused at 34C and ATP levels in the outflow of the chamber were measured using a bioluminescent assay. Illumination

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with 445 nm light at ~7 mW/mm2 evoked a +95% increase in ATP release (Figure 3E) (Gourine et al., 2010). This demonstrated directly that this vector is able to control astrocytic ATP release.

3.3 In vitro and in vivo application of AVV-sGFAP-ChR2(H134R)-Katushka1.3

In vitro AVV-sGFAP-ChR2(H134R)-Katushka1.3 has been used to study the role of astrocyte to neurone signalling in the retrotrapezoid nucleus (RTN) region. This nucleus is located in the lower brainstem and implicated in the process of central chemosensitivity which enables the brain to adjust respiratory activity to maintain stable levels of CO2 and pH (Loeschcke, 1982;Mulkey et al., 2004). We hypothesised that chemosensitive astrocytes in that area may be able to excite local RTN neurones via release of ATP (Figure 4A). We took advantage of the fact that RTN neurones can be selectively targeted with the PRSx8 promoter (Abbott et al., 2009). Two vectors were applied simultaneously to organotypic slices containing the RTN area, AVVsGFAP-ChR2(H134R)-Katushka1.3 for optogenetic control of astrocytes and AVV-PRSx8DsRed2 to label RTN neurones with DsRed2 for identification and patch clamp recordings. Stimulation of local astrocytes transduced with AVV-sGFAP-ChR2(H134R)-Katushka1.3 by flashing light evoked lasting depolarisations of patched DsRed2-labelled RTN neurones (Figure 4B). In the presence of the ATP receptor blocker, MRS2179 (10M), these depolarisations were reversibly prevented (Gourine et al., 2010), indicating a pivotal role for ATP in this process. In vivo, AVV-sGFAP-ChR2(H134R)-Katushka1.3 was used to test whether optogenetic excitation of local astrocytes in the RTN area can trigger a chemoreceptor-like response. In anaesthetised, vagotomized, and artificially ventilated rats pre-injected with vector 7-10 days prior to the experiment, the VS was exposed and phrenic nerve activity was recorded to monitor central respiratory drive. Unilateral illumination (pulsing 445 nm light from a laser source) of the transduced side of the VS evoked robust respiratory activity from hypocapnic apnea (Figure 4C). Furthermore, consistent with the outcome of in vitro experiments, application of MRS2179 (100M) reversibly inhibited the respiratory effects of optogenetic stimulation of astrocytes, confirming the involvement of ATP (Gourine et al., 2010). The intensity of light stimulation and the titres of viral vectors used in these experiments had to be carefully adjusted in order to achieve good reversibility and reproducibility of the physiological responses. Use of very high titres of vectors (>1011 transducing units/ml) resulted in over-expression and cell death.

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4. The delivery systems The studies outlined above illustrate the power of optogenetics for studies of astrocyte to neurone communication. The ability to target astrocytes selectively and moreover, at the same time target neurones with a different construct opens a tremendous window of opportunity for future research in this direction. These experiments demonstrate also the potential of cell-specific viral targeting as experimental strategy for neuroscience. For many of the cell types present in mammalian CNS there are fairly short and specific promoters which can be used in viral backbones and it may be predicted that in the future their arsenal will expand. The ability to selectively target two phenotypes (e.g. astrocytes and neurones) at the same time by simply mixing two viral vectors compares favourably in terms of speed, costs and flexibility to a protocol where a double transgenic has to be generated.

Many cell specific mammalian promoters are fairly weak compared to the non-selective viral promoters which are highly successful in cell lines. The promoter of the GFAP gene has been used for many years to target astrocytes and there are several transgenic mice with transgenes under control of this promoter (Pascual et al., 2005;Potokar et al., 2009). However, as mentioned previously, GFAP promoter sequences used for gene targeting have low transcriptional activity. Adeno-associated vectors partially off-set this problem by invading the target cells in very high copy numbers (Ortinski et al., 2010), but we choose to use AVV because of their important advantages for experimentation in slice cultures (high transducing efficiency in vitro, speed of expression, ease of preparation of large quantities), in addition to being very efficient in vivo (Teschemacher et al., 2005a), especially for targeting astrocytes (Duale et al., 2005). Transcriptional amplification strategy (TAS) can be used to enhance the activity of cell-specific promoters without loss of cell type specificity (Liu et al., 2008). For the development of the new viral vector we used the shortened GfaABC1D (694-bp) which has the same level of specificity as the longer versions of GFAP promoter (Su et al., 2004;Lee et al., 2008). A two step TAS was used to enhance transgene expression of GfaABC1D (Liu et al., 2008) (Figure 4D). TAS employs a minimal core promoter (65 bp) derived from the human cytomegalovirus (CMV) which is joined upstream of the GfaABC1D promoter in antisense orientation. Minimal CMV (mCMV) drives the expression of an artificial transcriptional enhancer (GAL4BDp65) (Liu et al., 2006) which then interacts with unique artificial binding sites (GAL4BS) upstream of the cell-specific promoter leading to an enhancement of the transcription of the ChR2(H134R)Katushka1.3 cassette (Figure 4D). Importantly, the mCMV-driven expression is still strongly

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influenced by the GfaABC1D promoter which in such constructs acts bi-directionally (Amendola et al., 2005). Therefore the enhancer expression is also selective to astrocytes. GAL4 binding sites do not exist in mammalian genome and this prevents off-target amplification and potential bias in gene expression. The TAS strategy is not restricted to the GFAP promoter and has been successfully used to amplify other cell-specific promoters in our laboratory, for example synapsin-1 (Liu et al., 2008) and tryptophan hydroxylase II (Benzekhroufa et al., 2009). We hope that its wider implementation will aid further design of highly efficient viral vectors for optogenetic experimentation.

5. Conclusion The development of optogenetic tools has made considerable progress in the last few years. This review has briefly summarised current data on the application of genetically encoded Ca2+ sensors and ChR2-derived light sensitive effectors to studies of astrocytes, thus illustrating how they could potentially aid in our understanding of astrocyte to neurone communication. Although biosensors have to yet outperform the best synthetic dyes, many applications in cell biology and physiology are only possible with GECI. One invaluable advantage of these indicators is the possibility to selectively express them in precise cell types which permits identity of the cellular source of the fluorescent signal in vitro and in vivo with confidence. The theoretical limits for GECI performance have not yet been reached and improvements certainly come with the advent of even brighter and possibly red-shifted FPs. This last point is particularly important because it will allow much deeper penetration into the brain tissue without the need of infra-red lasers and is expected to improve signal-to-noise levels. We have also reviewed the optogenetic approach to control astrocytic Ca2+ signalling and transmitter release in vitro and in vivo. Expressing ChR2(H1234R) selectively in astrocytes enabled us to trigger in these cells [Ca2+]i elevations, release of ATP, downstream events in RTN neurones, and increase in respiratory activity (Gourine et al., 2010).

As the field of optogenetics matures more tools transpire (Airan et al., 2009;Looser et al., 2009). These constructs are similar to ChR2 in that they are activated by light but, unlike ChR2, they impinge on the intracellular signalling rather than act via opening of ion channels in the plasma membrane. With the aid of these new tools, we shall have more ways to better understand how astrocytes signal to each other and to the surrounding neuronal networks.

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Acknowledgements SK, AGT, MF, BHL, JH, A.V.G., N.M and V.K. are supported by Wellcome Trust and British Heart foundation. SL is supported by BBSRC. DMC and EAS were supported by Molecular and Cell Biology program RAS.

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Figures Figure 1. A schematic representation of single and dual FP-based Ca2+ indicators. 1A: Layout of the cpGFP-based Ca2+ sensors, such as GCamps and Case12. CaM is the Ca2+ binding motif from calmodulin which binds to its target binding protein, M13 in the presence of Ca2+. These motifs are fused to the split GFP molecule with the linker peptide in the middle of it. Association of CaM and M13 is changing the configuration of the barrel, presumably making it tighter and this leads to an increase in fluorescence. 1B: Changes in fluorescence typical single-FP based Ca2+ indicators, such as Case12 or GCaMPs (left panel) and dual-FP based FRET indicators (right panel). The single FP-based indicator shows one emission peak in the presence of Ca2+, which is significantly increased in the presence of Ca2+ compared to 0 Ca2+ conditions in the presence of EGTA. The dual FP-based indicators have two emission peaks corresponding to the two FP and therefore require imaging into two separate channels. In the presence of Ca2+ FRET from blue-shifted protein to the red-shifted one leads to a change in the ration between the channels. 1C: Layout of cameleon-like FRET Ca2+ biosensors based on calmodulin-M13 interaction. Binding of Ca2+ by CaM causes it to bind its target protein M13 and brings CFP and YFP in a position favourable for FRET, resulting in an increase of the YFP/CFP fluorescence ratio. 1D: Schematic depiction of the proposed model for a FRET Ca2+ biosensor based on the troponin C backbone. Binding of Ca2+ by troponin C causes it to undergo a conformational change, leading to an increase in YFP/CFP fluorescence ratio. Most published studies recommend 430 nm light sources for excitation of these constructs or a use of multi-photon microscope. However, we have verified that the 456 nm line of argon laser present on all standard confocal microscopes (Leica and Ziess) is at least as efficient for imaging TN-XXL. Emission was sampled at 480-520 nm and 535-590 nm bands. Figure 2. Fluorescent changes in the GECI, Case12 demonstrates acidification-evoked [Ca2+]i responses in the ventral brainstem surface (VS) astrocytes. 2A: AVV-sGFAP-Case12 (Souslova et al., 2007) transduced VS astrocytes of organotypic brainstem slice. The yellow arrow shows the direction of the flow in the chamber. A pH change from 7.4 to 7.2 causes a significant increase in the fluorescent intensity of Case12, indicating an increase in [Ca2+]i. 2B: Case12 vs conventional Ca2+ indicator Rhod-2: (i) Case12 demonstrates that TTX (1 M) does not prevent acidification-induced Ca2+ excitation of VS astrocytes (slice preparation of an adult rat). (ii) In similar experiments astrocytes were imaged with Rhod-2. Note that the maximal increase in fluorescence intensity is similar with Case12 and Rhod-2. Note also that by the end of the application of the acidic solution Case12 fluorescence in (i) is reduced due to its reversible quenching. This is a common feature of all sensors based on cyclically permutated GFP.

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2C: Case12 fluorescent intensity in the presence of Ca2+ follows that of the conventional indicator Fura-2. Simultaneous imaging of pH- and ATP-evoked responses in the same VS astrocytes using Case12 and Fura-2 to confirm Ca2+ sensitivity and dynamic range of Case12 (dissociated VS astrocyte culture). Note that following application of the acidic solution Case12 fluorescence is reduced due to its reversible quenching. Here and in figures 3 and 4 some panels are modified from Gourine et al (2010.) Figure 3. Validation of ChR2-Katushka1.3 (currently known as Katushka 2) fusion of optogenetic control of astrocytes. 3A: Fluorescence spectra of Rhod-2 and Katushka1.3 are sufficiently well separated allowing Ca2+ imaging using Rhod-2 within the 570-610 nm band. While their spectra partially overlap, in practice this does appear to interfere with Ca2+ imaging (see Figure 3C for example). 3B: Optical path for confocal Ca2+ imaging and light activation of ChR2. Leica SP2 upright confocal microscope was modified as following: (i) the standard Hg lamp was replaced by a LED-based illumination system; (ii) an additional diachronic mirror was placed into the light path with the cut-off at 500 nm. This mirror reflected the light from the 470 nm LED onto the specimen while being transparent for both, the yellow 561 nm laser and the emitted red fluorescence. Thus, activation of ChR2 was possible while sampling Rhod-2 fluorescence with minimal interference. 3C: Light stimulation of the primary cultured astrocytes transduced with AVV-sGFAPChR2(H134R)-Katushka1.3 evokes significant [Ca2+]i responses in all the transduced astrocytes (2 mM external Ca2+, 470 nm light, 20/20 msec duty cycle). 3D: ChR2-induced [Ca2+]i increase in primary astrocytes partially depends on extracellular Ca2+. [Ca2+]i elevations were significantly reduced following 30 min of incubation in 0 Ca2+ media. 3E: ATP release following illumination of VS areas in organotypic brainstem slices (n=5) transduced with AVV-sGFAP-ChR2(H134R)-Katushka1.3. The RLU (relative luminescence unit) value is proportional to the ATP concentration. Figure 4. Optogenetic control of astrocytes in vitro and in vivo. 4A: Schematic of the experiment where light-induced activation of VS astrocytes in the RTN area triggers a respiratory response. Stimulated astrocytes confer excitation to the local RTN neurones which are coupled to the respiratory network to activate respiratory activity. 4B: ChR2-expressing astrocytes stimulated by blue light evoked strong depolarisations of the local RTN neurones. Depolarisations were recorded by patch clamp of DsRed2-labelled RTN neurones. Application of the ATP receptor blocker MRS2179 (10M), prevented these depolarisations, suggesting that ATP plays an important role in this process (not shown, see (Gourine et al., 2010).

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4C: Light stimulation of ChR2-expressing astrocytes of the brainstem VS evoked bursts of the respiratory activity in anaesthetised rats transduced with AVV-sGFAP-ChR2(H134R)Katushka1.3. Animals were hyper-ventilated to prevent natural rhythmic activity of the phrenic nerve. IPNA integrated phrenic nerve activity. Topical application of MRS2179 (100M) on the VS of the medulla eliminated this response. 4D: TAS illustrated using AVV-sGFAP-ChR2(H134R)-Katushka1.3 (later renamed Katushka 2). This approach allows strong enhancement of mammalian promoters with no loss of cell specificity.

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