Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging.DOI: http://dx.doi.org/10.7554/eLife.12727.001

Genetically-encoded calcium indicators (GECIs) facilitate imaging activity of genetically defined neuronal populations in vivo. The high intracellular GECI concentrations required for in vivo imaging are usually achieved by viral gene transfer using adeno-associated viruses. Transgenic expression of GECIs promises important advantages, including homogeneous, repeatable, and stable expression without the need for invasive virus injections. Here we present the generation and characterization of transgenic mice expressing the GECIs GCaMP6s or GCaMP6f under the Thy1 promoter. We quantified GCaMP6 expression across brain regions and neurons and compared to other transgenic mice and AAV-mediated expression. We tested three mouse lines for imaging in the visual cortex in vivo and compared their performance to mice injected with AAV expressing GCaMP6. Furthermore, we show that GCaMP6 Thy1 transgenic mice are useful for long-term, high-sensitivity imaging in behaving mice.

Small molecules are important tools to measure and modulate intracellular signaling pathways. A longstanding limitation for using chemical compounds in complex tissues has been the inability to target bioactive small molecules to a specific cell class. Here, we describe a generalizable esterase-ester pair capable of targeted delivery of small molecules to living cells and tissue with cellular specificity. We used fluorogenic molecules to rapidly identify a small ester masking motif that is stable to endogenous esterases, but is efficiently removed by an exogenous esterase. This strategy allows facile targeting of dyes and drugs in complex biological environments to label specific cell types, illuminate gap junction connectivity, and pharmacologically perturb distinct subsets of cells. We expect this approach to have general utility for the specific delivery of many small molecules to defined cellular populations. cellular imaging | microscopy | enzyme substrates | fluorophores | pharmacological agents C hemical probes are essential tools in biology for measuring and manipulating cellular properties. Optimization of the structural and electronic features of small molecules allows the fine-tuning of molecular recognition specificity for a particular cellular target. Even with high molecular specificity, however, the application of small molecules in complex biological environments is frequently limited by poor cellular specificity. The inability to target small molecules, such as imaging or pharmacological agents, to defined cellular populations can confound the evaluation and control of discrete subsets of cells within a multicellular environment. A general and efficient strategy for cell-specific targeting, combining the molecular specificity of small molecules with the cellular specificity of genetics, would allow intracellular pathways in defined cell types to be selectively probed in complex tissues.An attractive approach for general cell-specific delivery of small molecules employs selective enzyme-substrate pairs. In this strategy, compounds are masked by attachment of a standard, disposable blocking group that is stable to native cellular enzymes, but labile to a specific exogenous enzyme. Expression of such a protein in a genetically defined cell population permits unmasking of the small molecule with cellular specificity. To be useful across experimental paradigms, such a system should utilize an enzyme that unmasks molecules with high efficiency, expresses in different cell types, and exhibits low cellular toxicity. The cognate masking group must be modular, synthetically efficient, and allow molecules to diffuse passively across the cellular membrane, while also exhibiting favorable solubility and stability in aqueous solution.To date only a few enzyme-substrate pairs have been used as targeted delivery systems for small molecules, and none meet all the criteria outlined above. Strategies employing enzymes encoded by common reporter genes (1) have found some success in targeting small molecules (2-4), ...