ABSTRACT
Free calcium ions (Ca2+) are important and omnipresent second messengers for intracellular signal transduction with key regulatory functions in numerous biological processes. Ca2+ signals are involved in diverse processes ranging, for example, from gene expression, apoptosis and cell death, bacterial chemotaxis, cell division, and fertilization, to the activation of cells of the immune system. As such, Ca2+ signals are extremely versatile owing to their complex spatiotemporal regulation. In neurons, Ca2+ is among the most important intracellular signals driving essential mechanisms in neurobiological communication. Action potentials (APs) can trigger and release bursts of Ca2+ through voltage-gated channels, causing rapid changes in intracellular Ca2+ concentrations. Similarly, Ca2+ inuxes can be triggered by the release of neurotransmitters, leading to the activation of neurotransmitter-gated ion channels such as N-methyl-daspartate (NMDA) receptors (Müller and Connor 1991; Jae et al. 1992; Berridge et al. 2000). Notably, the processes regulated by Ca2+ signaling span a wide time
3.1 Introduction 73 3.2 Genetically Encoded Calcium Indicators 74 3.3 Strategies for Designing Genetically Encoded Calcium Indicators 77
3.3.1 Calmodulin-Based FRET Calcium Indicators 77 3.3.2 Troponin C-Based FRET Calcium Indicators 79 3.3.3 Single-Wavelength/Single Fluorescent Protein Indicators 80
3.4 FRET or Single-Wavelength Probes? 82 3.5 Biocompatibility, Expression Levels, and Buering of Cellular Calcium 83 3.6 Conclusions 85 References 85
frame from microsecond release of neurotransmitters in synapses to gene transcription lasting up to several hours (Berridge et al. 2003). e magnitude of activity-induced changes in neurons makes Ca2+ a unique ion to monitor neuronal activity (Hille 1992). Monitoring changes in free [Ca2+] in spines, dendrites, axons, or somas can be used as a reliable representation of neuronal activity by providing an indirect measure of action potential frequency (Denk et al. 1996; Svoboda et al. 1996, 1997). us, Ca2+ imaging has become an important method to report the activity of various neuronal cell types and to understand the connectivity and signaling pathways in neuronal networks.
