Genetically Encoded Optical Sensors for Monitoring of Intracellular Chloride and Chloride-Selective Channel Activity

This review briefly discusses the main approaches for monitoring chloride (Cl−), the most abundant physiological anion. Noninvasive monitoring of intracellular Cl− ([Cl−]i) is a challenging task owing to two main difficulties: (i) the low transmembrane ratio for Cl−, approximately 10:1; and (ii) the small driving force for Cl−, as the Cl− reversal potential (ECl) is usually close to the resting potential of the cells. Thus, for reliable monitoring of intracellular Cl−, one has to use highly sensitive probes. From several methods for intracellular Cl− analysis, genetically encoded chloride indicators represent the most promising tools. Recent achievements in the development of genetically encoded chloride probes are based on the fact that yellow fluorescent protein (YFP) exhibits Cl−-sensitivity. YFP-based probes have been successfully used for quantitative analysis of Cl− transport in different cells and for high-throughput screening of modulators of Cl−-selective channels. Development of a ratiometric genetically encoded probe, Clomeleon, has provided a tool for noninvasive estimation of intracellular Cl− concentrations. While the sensitivity of this protein to Cl− is low (EC50 about 160 mM), it has been successfully used for monitoring intracellular Cl− in different cell types. Recently a CFP–YFP-based probe with a relatively high sensitivity to Cl− (EC50 about 30 mM) has been developed. This construct, termed Cl-Sensor, allows ratiometric monitoring using the fluorescence excitation ratio. Of particular interest are genetically encoded probes for monitoring of ion channel distribution and activity. A new molecular probe has been constructed by introducing into the cytoplasmic domain of the Cl−-selective glycine receptor (GlyR) channel the CFP–YFP-based Cl-Sensor. This construct, termed BioSensor-GlyR, has been successfully expressed in cell lines. The new genetically encoded chloride probes offer means of screening pharmacological agents, analysis of Cl− homeostasis and functions of Cl−-selective channels under different physiological and pathological conditions.


INTRODUCTION
Fluorescent indicators designed for quantitative monitoring of intracellular ions and analysis of the distribution of various proteins have brought about a revolution in obtaining important information about the functioning, development and pathology of cells and cellular components of biological organisms.
In this review we will briefl y discuss the main approaches for monitoring chloride (Cl − ), the most abundant physiological anion. Cl − is present in every cell of biological organisms and participates in a variety of important cellular processes, such as neurotransmission, regulation of cell volume, pH and water-salt balance. The concentration of intracellular Cl − and its permeance is highly regulated by a variety of Cl − -selective channels and Cl − transporters Genetically encoded optical sensors for monitoring of intracellular chloride and chloride-selective channel activity resting potential of the cells. Consequently, sensitive probes with high dynamic range at physiological [Cl − ] i are necessary for reliable analysis of [Cl − ] i distribution and its functional variations.
For [Cl − ] i monitoring several methods have been proposed. The most used are Cl − -selective microelectrodes; chloride-sensitive fl uorescent dyes and genetically encoded chloride-sensitive probes. We will briefl y describe these approaches with the main focus on genetically encoded chloride-sensitive probes, which are the most promising tools for effective analysis of Cl − homeostasis in various cell types.

Cl − SELECTIVE MICROELECTRODES
In the 1960s, 70s and 80s the use of ion-selective electrodes was the main available technique for intracellular Cl − detection. It allowed valuable information on Cl − distribution and dynamics in a number of cell types of biological organisms to be obtained. In very early studies, AgCl electrodes were used as tools for [Cl − ] i estimation (Mauro, 1954;Keynes, 1963;Strickholm and Wallin, 1965;Sato et al., 1968). With an electrode consisting of a fi ne AgCl wire protruding from the end of a glass capillary, [Cl − ] i was measured in giant axons of squid (Mauro, 1954;Keynes, 1963) and crayfi sh (Strickholm and Wallin, 1965). However, later observations demonstrated that all microelectrodes that use AgCl as the sensitive element develop "the same type of error in the intracellular environment and thus all give erroneously high values of [Cl − ] i " (Neild and Thomas, 1974). The improved method was based on the use of siliconized borosilicate glass micropipettes, the tips of which were fi lled with liquid chloride ion exchanger. This technique was introduced by Walker (1971) and used with some modifi cations in a number of studies Neild and Thomas, 1974;Ascher et al., 1976;Vaughan-Jones, 1979). These electrodes ( Figure 1A) had a small tip (1-2 µm) and gave complete responses to changes in Cl − within 1-2 min. Preparing these electrodes is a time-consuming procedure and penetration of cells without damage is diffi cult.
An intracellular Cl − -sensitive microelectrode records the algebraic sum of the membrane potential (E m ) and a voltage proportional to changes in Cl − activity. It means that E m must be separately determined using an independent electrode ( Figure 1B). To diminish the damage from insertion of two microelectrodes into a single cell, double-barrelled pipettes were proposed (Aickin, 1981) and used for monitoring the intracellular Cl − activity in smooth muscle cells (Aickin and Brading, 1982;Davis et al., 2000), retinal pigment epithelium (Bialek et al., 1995;La Cour et al., 1997) and in other cell types (Kitano et al., 1995;Debellis et al., 2001;Ianowski et al., 2002).
Early studies with Cl − -sensitive microelectrodes already demonstrated that [Cl − ] i in cells differs substantially from a predicted passive distribution, suggesting that Cl − ions must be actively transported through cellular membranes. These observations were confi rmed by the more recent discovery of several mechanisms of transmembrane Cl − transport (rev. Russell, 2000;Lauf and Adragna, 2004).
Three main obstacles limited the spread of the Cl − -sensitive microelectrode technique: (i) time-consuming procedure for microelectrode preparation; (ii) slow kinetics ( Figure 1C); and (iii) the need to use relatively large cells for reliable recording without cell damage. In addition, penetration of cells could change the native intracellular Cl − distribution. Methods based on imaging techniques are more promising as they provide an opportunity to monitor Cl − activity noninvasively and in populations of cells.

FLUORESCENT Cl − − SENSITIVE DYES
Because of the possibility of monitoring noninvasively the distribution and dynamics of ion concentration changes, fl uorescent Cl −sensitive dyes are the most popular approach for analysis of Cl − and Cl − -dependent physiological processes in different cells types.
The fl uorescence of many fl uorophores is decreased on application of heavy-atom anions, such as bromine and iodine. Cl − ions are less effective in this respect and relatively few fl uorophores are quenched by Cl − (Geddes et al., 2001). The background for Cl − monitoring was established by George Stokes, who described in 1852 the phenomenon of fl uorescence. In 1869 Stokes observed that the "fl uorescence of quinine in dilute sulfuric acid was reduced after the addition of hydrochloric acid, i.e., chloride ions" (Geddes et al., 2001). Perhaps the fact that quinine, which is sensitive to chloride, contains a quinoline ring has stimulated the production of many quinoline analogues in the search for effi cient Cl − probes.
The major disadvantage of these probes comes from their spectral properties, i.e. excitation at ultraviolet wavelengths. As a result, they are prone to strong bleaching (Inglefi eld and Schwartz-Bloom, 1997;Nakamura et al., 1997, Figure 2B). This restricts the duration of the measurements and allows only a very low data acquisition rate (0.2-2 frames per minute) (Inglefi eld and Schwartz-Bloom, 1997;Fukuda et al., 1998;Sah and Schwartz-Bloom, 1999). However, the use of these dyes in combination with two-photon microscopy strongly reduces bleaching and, consequently, photochemical damage (Marandi et al., 2002;Funk et al., 2008).

GENETICALLY ENCODED Cl − INDICATORS YFP AS A TOOL FOR INTRACELLULAR Cl -MONITORING
An alternative to exogenously added indicators is the use of an endogenously expressed chromophore such as green fl uorescent protein (GFP). Using appropriate targeting sequences, GFPs have been directed selectively to numerous intracellular sites. Various applications of GFP in physiological studies of living cells have been described Zaccolo and Pozzan, 2000;Bizzarri et al., 2009). GFP derivatives with different colors have been used in FRET models to monitor Ca 2+ (Miyawaki et al., 1997(Miyawaki et al., , 1999, pH (Kneen et al., 1998;Llopis et al., 1998;Miesenbock et al., 1998) and protein-protein interactions (Heim, 1999). During the last decade, a new method of noninvasive [Cl − ] i monitoring using genetically encoded optical probes has been developed. This approach is based on the halide-binding properties of yellow A relation between the Cl − concentration and the fl uorescence intensity is described by the Stern-Volmer-equation: where F 0 is the fl uorescence in the absence of halide, F is the fl uorescence in the presence of halide and K SV is the Stern-Volmer quenching constant (in M −1 ). From this equation the EC 50 , the concentration of Cl − causing a 50% decrease in fl uorescence, is 1/K SV . Table 1 shows values of the Stern-Volmer constant and corresponding EC 50 values for some quinolinium indicators. The mostused quinolinium indicators are 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), 6-methoxy-N-ethylquinolium (MEQ) and N-(6-methoxyquinolyl)-acetoethyl ester (MQAE).
The fi rst designed quinolinium-based Cl − indicators was SPQ (Wolfbeis and Urbano, 1983;Illsley and Verkman, 1987; Figure 2A, Table 1). SPQ is excited at ultraviolet wavelengths with absorbance maxima at 318 nm and 350 nm. This fl uorescent dye has a single broad emission peak centred at 450 nm with a quantum yield of 0.69 in the absence of halides. In aqueous buffers the Stern-Volmer constant for quenching of SPQ by Cl − is 118 M −1 , giving EC 50 ∼ 8.5 mM. The fl uorescence of SPQ is not altered by cations, phosphate, nitrate or sulfate, but it is quenched weakly by other monovalent anions including citrate, acetate, gluconate and bicarbonate (Illsley and Verkman, 1987;Krapf et al., 1988b;Jayaraman et al., 1999).
MQAE, the other quinolinium-based dye, which has been used in a number of studies during recent years, has high Cl − sensitivity (Figure 2A, Table 1) and, unlike SPQ, easily permeates through the plasma membrane (Mansoura et al., 1999). As a result the time of incubation with this dye can be rather short. For instance, 10-min incubation of brain slices is suffi cient for bright staining of neurons (Marandi et al., 2002). However, this substance has to be used with prudence, as incubation of slices for 30-45 min caused a deterioration in the properties and even death of many neurons in neocortex and hippocampal slices (Holmgren, Zilberter, Mukhtarov, personal observations). For these reasons, results obtained after as long as 1-2 h of treatment with MQAE (e.g. Servetnyk and Roomans, 2007) have to be interpreted with caution. The other disadvantages of this dye lie in the signifi cant leakage rate and bleaching. The leakage rate seems to be preparation-specifi c, ranging from 3% per hour fl uorescent protein (YFP) and it derivatives . YFP is a derivative of GFP, which contains four point mutations (T203Y/S65G/V68L/S72A). It has improved brightness and red-shifted excitation/emission spectra compared with GFP (Ormo et al., 1996;Elsliger et al., 1999). The halide sensitivity of YFP was conferred on this protein using a rational mutagenesis strategy based upon crystallographic data (Wachter et al., 1998) and confi rmed experimentally . It was found that YFP fl uorescence is sensitive to various small anions with relative potencies . YFP sensitivity to these small anions results from ground-state binding near the chromophore , which apparently alters the chromophore ionization constant and hence the fl uorescence emission. As with other GFP derivatives, the fl uorescence of YFP is pH-dependent. The EC 50 values for Cl − varied from 32.5 mM (pH = 6) to 777 mM (pH = 7.5) ( Table 2).
This analysis demonstrates that at the physiological range of intracellular pH (7.2-7.4) the sensitivity of YFP to Cl − is low, which creates diffi culties and limitations in using "wild type" (WT) YFP as a Cl − indicator. Indeed, different methods of [Cl − ] i estimation in various cell types gives its range of variations from 3 to 60 mM (Table 3). Consequently, the resolution of the indicator with EC 50 more then 100 mM is low and it can lead to errors in noninvasive estimation of [Cl − ] i .
To further improve spectral characteristics, YFP was subjected to additional mutagenesis and the most successful variants were selected. It has been demonstrated that at pH 7.5 the EC 50 for Cl − of the mutant YFP H148Q is 154 mM ( Table 2; Wachter et al., 2000), which is still high; however, it is closer to the physiological range of [Cl − ] i than WT YFP (777 mM).
To enhance sensitivity of YFP-H148Q to Cl − , libraries of mutants were generated in which pairs of residues in the vicinity of the halide binding site were randomly mutated (Galietta et al., 2001a). Analysis of over a thousand clones revealed improved anion sensitivity with EC 50 s down to 40 mM for Cl − (V163S), 10 mM for NO 3 − (I152L) and 2 mM for I − (I152L). To check physiological applicability, the I152L mutant, which exhibited the best I − and NO 3 − sensitivities, was expressed in Swiss 3T3 fi broblasts carrying CFTR. Transfected cells were brightly fl uorescent with a uniform cytoplasmic and nuclear staining pattern. Replacement of 20 mM Cl − with I − produced a fl uorescence decrease of 53 ± 2% with YFP-I152. It was much greater than that of <10% for the same experiment with YFP-H148Q, indicating that this mutant is a good tool for monitoring I − and NO 3 − . The same results were obtained when the activity of CFTR was studied using a Cl − /NO 3 − exchange protocol (Galietta et al., 2001b). Some other mutants of YFP also showed greatly improved Cl − sensitivities (Table 4), which stimulated further development of Cl − indicators. It provided the basis for using YFP mutants as genetically encoded Cl − sensors that can be targeted to specifi c organelles in living cells or expressed in specifi c cell types for monitoring [Cl − ] i distribution, to study the functioning of Cl − channels and pumps.

APPLICATION OF YFP IN HIGH-THROUGHPUT SCREENING
In a number of experimental models YFP derivatives have been used as suitable probes for high-throughput (HTP) screening. These allow the testing of tens of thousands of different compounds (Ma et al., 2002a,b). We present here several examples. YFP-H148Q was transfected in Fisher rat thyroid cells (FRT) and in Swiss 3T3 fi broblasts for quantitative HTP screening of potential modulators of CFTR halide permeances (Galietta et al., 2001b).
A mutant with enhanced sensitivity to halides (YFP-V163S) has been used in M1 cortical collecting-duct cells to monitor changes in Cl − mediated by CFTR or by stimulation with cAMP-and Ca 2+increasing agonists (Adam et al., 2005). Important observations have been obtained with YFP-H148Q/ I152L, which shows a 30-fold selectivity to I − over Cl − . It was proved to be a sensitive biosensor of Na + /I − symporter-mediated I − uptake in thyroid cells and nonthyroidal cells following gene transfer (Rhoden et al., 2007(Rhoden et al., , 2008. As defective iodide transport occurs in several inherited and acquired thyroid disorders, using this YFP mutant for detection of I − represents a useful tool for studying the pathophysiology and pharmacology of this Na + /I − symporter (Rhoden et al., 2007).
YFP derivatives were also used for testing ligands of glycine receptors (GlyRs) and ionotropic GABA receptors. Using HEK 293 cells expressing YFP-I152L or YFP-V163S mutants with these Cl − -selective receptor-operated channels has established the optimal conditions for pharmacological screening of Cl − (Kruger et al., 2005) and detection of functional and non-functional mutations in the GlyRs (Gilbert et al., 2009).
These observations have demonstrated advantages of using genetically encoded YFP derivatives in HTP in comparison with other techniques.

ADVANTAGES AND DISADVANTAGES OF GENETICALLY ENCODED Cl − INDICATORS
Of the many advantages of YFP-based Cl − indicators in comparison with fl uorescent dyes, we will mention only the four most important. First, the peak of absorbance is at a wavelength of more then 480 nm, i.e., in contrast to quinolinium-based halide indicators, YFP can be excited in the visible range, permitting more stable fl uorescence signal and less cell damage. Consequently, it allows longlasting Cl − monitoring with repetitive stable responses and bright fl uorescence signals using conventional imaging equipment.
Second, genetically encoded probes can be targeted to specifi c cell types by cell-specifi c promoter, or to defi ned cellular compartments by fusion to short sequence tags or to specifi c proteins. This would allow Cl − monitoring in specifi c cell types or cellular compartments. For instance, transgenic mice expressing enhanced YFP (EYFP) under control of the Kv3.1 K + channel promoter (pKv3.1) have been generated (Metzger et al., 2002), making possible neuron-specifi c expression of EYFP in the hippocamus, thalamus and granule cell layer of cerebellum. This model has been used for analysis of glutamateinduced changes in intracellular Cl − and pH (Metzger et al., 2002;Slemmer et al., 2004). The thy1 promoter has been successfully used to drive specifi c neuronal expression of Clomeleon in the hippocampus and in neocortical areas as well as in the dentate gyrus, cerebellar mossy fi bers and piriform cortex (Berglund et al., 2008).
Third, the intracellular concentration of the YFP-based Cl − indicators is only a few micromolar, which is several orders of magnitude less than the [Cl − ] i . This avoids buffering effects, which are a substantial problem in, for instance, fl uorescence measurements of intracellular Ca 2+ at using conventional fl uorescent dyes.
Finally, the molecular weight of the YFP-based Cl − indicators is about 27 kDa, which prevents diffusion of the indicator from cells. In cells that are imaged without simultaneous electrophysiological recordings, indicator levels remains constant over hours.
YFP-based sensors also have several disadvantages. One of them is pH sensitivity ( Table 2). Changes in intracellular pH or in specifi c compartments can lead to errors in observations and interpretation of results. To overcome this problem, in some cases independent monitoring of pH is necessary.
YFP-based Cl − sensors have rather low kinetics of Cl − association/ dissociation. The double mutant YFP-H148Q/V163S, which exhibits relatively high Cl − sensitivity (EC 50 = 39 mM), has an association time constant τ = 1900 ms (Galietta et al., 2001a), which would cause limitations in the time resolution when using this mutant for analysis of rapid Cl − dynamics. The other double mutant, YFP-H148Q/I152L, has much faster association/dissociation kinetics (association time constant τ = 52 ms; Galietta et al., 2001a); however, its sensitivity to Cl − is relatively low (EC 50 = 85 mM). Thus, the YFP-based probes can be used to reliably detect changes in Cl − concentrations with time course resolution in the range of hundreds of milliseconds or even seconds. A signifi cant limitation in the use of Cl − indicators was a lack of a Cl − -dependent change in spectral shape, which precludes ratiometric measurements. For quinolinium-based Cl − -sensitive dyes, synthesis of a series of dual-wavelength fl uorescent indicators has been achieved using conjugation of Cl − -sensitive and Cl − -insensitive dye molecules with different spacers (Jayaraman et al., 1999). Only one "chimera" (MQa4AQ) was cell-permeating and it turned out to be four times less Cl − -sensitive than MQAE. YFP-derivatives also do not have a clear isosbestic point in spectral shape at different Cl − concentrations. This precludes ratiometric measurements and, consequently, gives rise to limitations in the estimation of [Cl − ] i values.
The important development of genetically encoded Cl − probes was achieved by Kuner and Augustine (2000), who constructed a ratiometric YFP-based Cl − indicator, termed Clomeleon.
The work of this probe is based on the phenomenon of Fluorescence Resonance Energy Transfer (FRET) between two fl uorescent proteins (see Box 1). Binding of a Cl anion to TFP reduces its emission, leading to a decrease in FRET effi ciency. This process can be visualized as a reduction in the ratio of fl uorescence emission between the TFP acceptor and CFP donor fl uorophores.
Analysis of emission spectra of this construct revealed that the intensity of fl uorescence depends on Cl − concentration. Moreover, presence of the isosbestic point in normalized spectra (Figure 3C) allows the use of this indicator as a ratiometric probe for estimation of Cl − concentration ( Figure 3D) The construct was named Clomeleon as an allusion to the FRET-based genetically encoded Ca 2+ indicator, Cameleon (Miyawaki et al., 1997).
Unlike organic probes, Clomeleon possesses several valuable features: excitation at visible wavelengths, good signal-to-noise ratio, safer loading procedures for cells, absence of leakage from cells and the possibility of targeting the probe to different cell types using specifi c promoters. Moreover, the construct exhibits high fl uorescence stability: absence of Clomeleon bleaching during 2 h of recording has been reported (Pond et al., 2006). Proteolitical stability of Clomeleon in transgenic mice has been observed during 9 months. It is also a low toxicity probe, which did not cause any behavioural aberration in Clomeleon-expressing mice in the course of 2 years .
The main advantage of Clomeleon is the possibility of performing ratiometric measurements of [Cl − ] i . The ratiometric capabilities of Clomeleon allow optical measurements that are minimally infl uenced by the thickness of the specimen, intensity of the excitation light and concentration of the indicator. This, in turn, makes it possible to accurately determine Cl − values even in cells with complicated geometry, such as neurons.
Clomeleon has been used for measurements of [Cl − ] i in cultured hippocampal neurons (Kuner and Augustine, 2000), in plant cells (Lorenzen et al., 2004) and in cells of retina and brain slices (Duebel et al., 2006;Pond et al., 2006). The widest fi eld Genetically encoded chloride sensors of application for genetically encoded probes comes from the possibility of targeting them to specifi c cell types using unique promoters, or to cellular compartments and membrane domains by fusion to respective tags or to proteins with known location. Several transgenic mouse lines have been created by insertion of Clomeleon, under control of the thy1 promoter, into their genome (Berglund et al., 2008). Details and functional implications of these models are described in recent papers (Duebel et al., 2006;Berglund et al., 2008). Potential limitations of this probe are that it, as other YFP-based Cl − indicators, is sensitive to pH and that the time course of reaction to Cl − is relatively slow. The other potential problem is that the fl uorophores, CFP and TFP, may bleach at different rates, which would distort the calibration of the indicator signal.
The important disadvantage of Clomeleon is that at physiological pH it has a rather low sensitivity to Cl − . The EC 50 of Clomeleon is more than 160 mM (Kuner and Augustine, 2000; Figure 3D, Table 5) which is far from physiological [Cl − ] i (3-60 mM). For this reason, the development of ratiometric probes with high sensitivity to Cl − is required.

Cl-Sensor
Recently a new genetically encoded indicator, termed Cl-Sensor, has been proposed ; Figure 4A). Analysis of the spectral properties of this construct during simultaneous monitoring of fl uorescence signals and whole-cell recordings with different Cl − concentrations in the pipette solution revealed two important features.
First, the normalized excitation spectra obtained at different [Cl − ] i have a common point near 465 nm (Figure 4B), meaning that Cl-Sensor allows ratiometric monitoring using the fl uorescence excitation ratio. This feature allows recordings using conventional setups with devices for a rapid change of excitation wavelength.
Second, due to triple YFP mutation (YFP-H148Q/I152L/V163S) this construct exhibits a relatively high sensitivity to Cl − with an estimated EC 50 of about 30 mM (28 ± 5 mM). With about 5-fold higher sensitivity than Clomeleon, this indicator has a good dynamic range at physiological intracellular concentrations (Figure 4C), providing a good basis for reliable monitoring of [Cl − ] i in different cell types.
Cl-Sensor demonstrates the same advantageous features as Clomeleon, i.e. excitation at visible wavelengths, good signal-to-noise ratio, safer loading procedures, absence of leakage from cells and ability to be targeted to different cell types using specifi c promoters.
Similarly to other fl uorescent proteins from the GFP family, Cl-Sensor exhibits a relatively high pH sensitivity with pKa ranging from 7.1 to 8.0 pH units at different Cl − concentrations.
One widespread problem with GFPs is their low transfection effi ciency in neurons. To overcome this diffi culty, Cl-Sensor was subcloned at two different vectors driven by mutated CMV and ubiquitine promoter. These plasmids carrying Cl-Sensor reveal higher transfection effi ciency and brightness of probe in the CHO cell line, retinal cells and spinal or hippocampal neurons (Waseem et al., paper in preparation).
The Cl-Sensor was used for noninvasive estimation of [Cl − ] i in CHO cells, hippocampal neurons and photoreceptor cells from retinal slices ( Table 3; Markova et al., 2008;Mukhtarov et al., Phenomenon of fl uorescence resonance energy transfer (FRET) represents interaction between two fl uorophores, when excitation energy from a donor (D) molecule is directly transferred to a molecule of acceptor (A).
Four main conditions have to be fulfi lled for this phenomenon development: (i) overlapping of emission spectrum of donor and excitation spectrum of acceptor; (ii) small distance between molecules (less then 10 nm); (iii) good orientation the dipole moments of donor emission and acceptor absorption; (iv) high quantum yield of fl uorophores.
The FRET effi ciency (E) is dependent on the inverse sixth power of the distance between fl uorophores (r): where R 0 is the distance at which the energy transfer effi ciency is 50%.
This makes FRET technique a sensitive tool for analysis of proteinprotein interaction and changes in intermolecular distances.
For more details see: Tsien et al., 1993;Pollok and Heim, 1999;Jares-Erijman and Jovin, 2003;Sekar and Periasamy, 2003;Wallrabe and Periasamy, 2005. BOX 1 | FRET.  (Matsuda and Cepko, 2004;Mukhtarov et al., 2008). The effi ciency of electroporation into the developing postnatal retina (at P0) was high, and transgene expression persisted for more then 1 month , indicating that in vivo electroporation of Cl-Sensor cDNA is a powerful tool for monitoring [Cl − ] i under different experimental conditions and through age-dependent changes in Cl − in neurons.

BioSensor-GlyR -tool for monitoring Cl − -selective channel activation
Investigation of brain functioning requires methods allowing dynamic analysis of network activity combined with determination of single-cell properties. This strategy has been developed for monitoring calcium transients using rapid two-photon microscopy (Cossart et al., 2005). Chemically engineered proteins that are directly sensitive to light are also powerful optical methods of protein function control for modulation of signalling circuits inside cells and in cell circuits (Gorostiza and Isacoff, 2008). However, analysis of networks formed by neuronal circuits for specifi c synapses (glutamatergic, GABAergic or glycinergic) is hampered by lack of adequate techniques. This problem could be solved by genetic incorporation of molecules capable of changing their fl uorescence on activation of specifi c synapses. The best candidates for these molecules would be fl uorescently modifi ed postsynaptic receptor-operated channels. Genetic incorporation of a molecular domain which could change fl uorescence upon channel activation would provide the possibility of noninvasive monitoring of ion channel activity. Development of these molecules is a highly challenging task. One of the approaches uses the voltage-clamp fl uorometry (VCF) technique, based on covalently attaching a small environmentally sensitive sulfhydryl-labeled fl uorophore to a cysteine introduced into a domain of interest on the protein. This approach has been successfully used to analyze the conformational rearrangements underlying gating of voltage-gated potassium channels (Mannuzzu et al., 1996) and ligand-gated glycine receptor (GlyR) channels Lynch, 2008, 2009).
The other way consists of inserting a genetically encoded fl uorescent sensor in the protein's sequence without changing its functional properties. Recently, a new genetic probe, termed BioSensor-GlyR, has been developed . This construct is a Cl − -selective GlyR channel with Cl-Sensor incorporated into the long cytoplasmic domain (Figure 5A).
The functioning of this modifi ed GlyR is not perturbed by the inserted Cl-Sensor. This fact was proved in whole-cell recordings of cells expressing BioSensor-GlyR, where rapid application of glycine elicited ionic currents with kinetics similar to those of wild-type GlyR ( Figure 5B). The main functional properties of BioSensor-GlyR, i.e. kinetics, agonist sensitivity and Cl − selectivity, are also similar to those of the wild-type GlyR.
Application of glycine to cells expressing BioSensor-GlyR induced changes in fl uorescence ( Figure 5C). The amplitude and direction of fl uorescence signals correlated with the amplitude and direction of glycine-induced currents (Figure 5D), demonstrating that BioSensor-GlyR is a good probe for spectroscopic monitoring of GlyR activation in live cells. The sensitivity of BioSensor-GlyR was high enough to resolve changes in [Cl − ] i induced by activation of postsynaptic receptors in glycinergic synapses. The decay kinetics of fl uorescence responses were slow compared with those of ionic currents. This might be partially due to the kinetics of Cl − binding by YFP. However, this may also refl ect slow intracellular Cl − transients, as monitoring with MQAE, the rapid quinolinium indicator, showed a similarly slow rise and decay of Cl − -dependent fl uorescence in cerebellar neurons (see Figure 2C).
In spite of these limitations, BioSensor-GlyR is a promising tool for spectroscopic monitoring of [Cl − ] i changes in the local surroundings of glycine receptor ion channels. Development of Genetically encoded chloride sensors transgenic animals expressing Cl-Sensor and BioSensor-GlyR will be particularly useful for studies of inhibitory neuronal networks in brain slice preparations using two-photon microscopy. For the Biosensor-GlyR it might, however, be a diffi cult task as expression of the GlyR, containing an additional CFP-YFP module in the long cytoplasmic loop, may modify the function of glycinergic synapses.

CONCLUSIONS
The development of imaging techniques and specifi c genetically encoded chloride-sensitive probes has opened new avenues for noninvasive monitoring of this ion in different cell types and cellular compartments in normal and pathological conditions. The main steps stimulating the development of these probes were as follows. First, the discovery of the halide sensitivity of YFP. Second, the production of YFP mutants exhibiting usefully high sensitivity to Cl − at concentrations close to the physiological range of [Cl − ] i . Third, the construction of molecules (Clomeleon and Cl-Sensor) consisting of two fl uorescent proteins, allowing ratiometric noninvasive estimation of [Cl − ] i . Finally, the incorporation of Cl-Sensor into the sequence of the Cl − -selective glycine receptor channel (BioSensor-GlyR), which opens up the ability to monitor synaptic activation of these proteins using imaging techniques. This provides an intriguing background for the development of biosensors which make it possible to monitor the activity of ionotropic GABA receptors and other Cl − -selective channels.