Genetically encoded calcium indicators.

One of the greatest challenges in neuroscience has been to monitor electrical activity and biochemistry in populations of identified neurons in ViVo. Recent work on new microscopy techniques has moved the field considerably further in that direction. In particular the combination of modern imaging technology and genetic labeling methods heralds a bright future for neuronal circuit analysis. Biosensor development complements these efforts on the “indicator side” by providing probes for key events crucial for an understanding of neuronal function and plasticity. Moreover it aims at overcoming long-standing limitations in the ability to monitor neuronal activity and biochemistry in intact tissues and over longer periods of time. The term “genetically encoded” refers to the fact that the sensors are solely composed of amino acids with no addition of any synthetic compound or cofactor that would be difficult to apply into a living brain. Thus, sensors are encoded by a stretch of DNA that can be manipulated, mutated, and concatenated by any technique that the recombinant DNA toolbox offers. When DNA coding for the sensor is delivered into a cell, the sensor is formed within the cell in situ. Combined with the use of cell-type specific promoters, cellular targeting sequences, and transgenic technology, this offers a noninvasive means to implant an indicator deeply within a tissue of a living organism with cellular and subcellular specificity. Genetically encoded indicators use the Green Fluorescent Protein (GFP) or more generally speaking autofluorescent proteins (XFPs) and mutants thereof as fluorophores with all the advantages and restrictions that these fascinating proteins possess. Currently the XFPs are the only known family of proteins that form an internal fluorophore in aerobic environments via an autocatalytic reaction involving a number of critical amino acid residues within the peptide backbone. Therefore progress on the biosensor side is closely linked with further improvements in engineering XFPs for more brightness, photostability, full maturation at 37 °C, and shifts to longer wavelengths of emission. In its strictest sense the term “genetically encoded calcium indicators” (GECIs) also includes other types of sensors that operate with chemiluminescence, such as aequorin and derived sensors that use chemiluminescence energy transfer from aequorin to GFP. While offering some advantages compared to fluorescent reporters such as lack of background fluorescence and the ease of working with freely moving animals, the difficulty of obtaining high spatial resolution makes these sensors less suitable for many brain imaging applications. As the topic is relatively large, these probes will not be covered here in more detail. Instead the reader is invited to consult other reviews. Traditionally optical measurements of free cytosolic calcium fluctuations have been perfomed using a collection of synthetic organic molecules that change fluorescence or absorbance properties upon calcium binding. The field was revolutionized by the work of Roger Y. Tsien, who generated a series of fluorescent polycarboxylate compounds with strongly improved fluorescence properties. Both singlewavelength indicators and indicators suitable for ratiometric imaging were established. Tsien also introduced noninvasive methods of loading the compounds into living cells as acetoxymethyl esters. During the last two decades improvements in design and performance were incorporated. Currently, the best dyes are characterized by large fractional fluorescence changes (for example an approximately 14-fold relative intensity change for the indicator Oregon Green BAPTA-1; www.invitrogen.com), very good selectivity for calcium versus other cations, fast binding and dissociation kinetics, fairly linear response properties, pH-resistance, and photostability. Thus, synthetic fluorescent calcium probes provide a standard of performance parameters that genetically encoded calcium indicators will be compared with. Measurements of free cellular calcium have received special interest in the neurosciences, because the geometry and high degree of compartmentalization of neurons, together with an exquisite collection of calcium channels specific to neurons, has added another twist to the already ravishing complexity of cellular calcium handling and signaling. Moreover, rises in cytosolic calcium can be used as a relatively direct measure of neuronal activity. When a neuron fires action potentials, voltage-gated calcium channels in the plasma membrane open up and lead to a rise in cytosolic calcium within a few milliseconds. Due to the relatively large * To whom correspondence should be addressed. E-mail: griesbeck@ neuro.mpg. de. Chem. Rev. 2008, 108, 1550–1564 1550