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 (E_Cl) 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.

Background and Purpose: Anion‐selective Cys‐loop receptors (GABA and glycine receptors) provide the main inhibitory drive in the CNS. Both types of receptor operate via chloride‐selective ion channels, though with different kinetics, pharmacological profiles, and localization. Disequilibrium in their function leads to a variety of disorders, which are often treated with allosteric modulators. The few available GABA and glycine receptor channel blockers effectively suppress inhibitory currents in neurons, but their systemic administration is highly toxic. With the aim of developing an efficient light‐controllable modulator of GABA receptors, we constructed azobenzene‐nitrazepam (Azo‐NZ1), which is composed of a nitrazepam moiety merged to an azobenzene photoisomerizable group.Experimental Approach: The experiments were carried out on cultured cells expressing Cys‐loop receptors of known subunit composition and in brain slices using patch‐clamp. Site‐directed mutagenesis and molecular modelling approaches were applied to evaluate the mechanism of action of Azo‐NZ1.Key Results: At visible light, being in trans‐configuration, Azo‐NZ1 blocked heteromeric α1/β2/γ2 GABA_A receptors, ρ2 GABA_A (GABA_C), and α2 glycine receptors, whereas switching the compound into cis‐state by UV illumination restored the activity. Azo‐NZ1 successfully photomodulated GABAergic currents recorded from dentate gyrus neurons. We demonstrated that in trans‐configuration, Azo‐NZ1 blocks the Cl‐selective ion pore of GABA receptors interacting mainly with the 2′ level of the TM2 region.Conclusions and Implications: Azo‐NZ1 is a soluble light‐driven Cl‐channel blocker, which allows photo‐modulation of the activity induced by anion‐selective Cys‐loop receptors. Azo‐NZ1 is able to control GABAergic postsynaptic currents and provides new opportunities to study inhibitory neurotransmission using patterned illumination.

Optogenetic and photopharmacological tools to manipulate neuronal inhibition have limited efficacy and reversibility. We report the design, synthesis, and biological evaluation of Fulgazepam, a fulgimide derivative of benzodiazepine that behaves as a pure potentiator of ionotropic γ‐aminobutyric acid receptors (GABA_ARs) and displays full and reversible photoswitching in vitro and in vivo. The compound enables high‐resolution studies of GABAergic neurotransmission, and phototherapies based on localized, acute, and reversible neuroinhibition.

Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cl_i]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cl_i]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC₅₀ for Cl_i was 46 mM, indicating that this line is appropriate for measuring neuronal [Cl_i] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC₅₀ for Cl_i was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cl_i in specific cell types in vivo.