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Fluorescent calciumsensors arewidely used to imageneural activity.Using structure-basedmutagenesis andneuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies andmice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials inneuronal somata and orientation-tuned synaptic calciumtransients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5-40-mm long).GCaMP6sensors thusprovidenewwindowsintothe organization anddynamics of neural circuitsovermultiple spatial and temporal scales.
Neural activity causes rapid changes in intracellular free calcium1-4. Calcium imaging experiments have relied on this principle to track the activity of neuronal populations5,6 and to probe excitation of small neurons and neuronal microcompartments2,7-10.Genetically encoded protein sensors can be targeted to specific cell types2,9,11,12 for non-invasive imaging of identified neurons and neuronal compartments8,13-15 over chronic timescales6.
Calcium indicator proteins include the single fluorophore sensor GCaMP (refs 11, 16, 17) and several families of Förster resonance energy transfer based sensors18-22. However, none of these proteinbased indicators have yet surpassed the sensitivity and speed of commonly used synthetic calcium indicators (for example, Oregon Green Bapta-1- AM,OGB1-AM). Therefore, depending on the experimental goals, investigators choose between sensitive synthetic indicators delivered by invasive chemical or physical methods, or less sensitive protein sensors delivered by genetic methods.
Multiple rounds of structure-guided design have made GCaMPs the most widely used protein calcium sensors11,16,17. But past efforts in optimizing GCaMPs and other indicators of neuronal function were limited by the throughput of quantitative and physiologically relevant assays. Because neurons have unusually fast calcium dynamics and low peak calcium accumulations4, sensors designed to probe neuronal function are best tested in neurons11,13,23,24, rather than in non-neuronal systems, most of which showmuch slower and larger calcium changes19. We thus screened GCaMP variants produced by mutagenesis in neurons, and subsequently validated lead sensors in several in vivo systems.
GCaMP protein engineering
GCaMP (ref. 17) and its progeny11,16 consist of circularly permuted green fluorescent protein (cpGFP)25, the calcium-binding protein calmodulin (CaM) and CaM-interacting M13 peptide26 (Fig. 1a). The CaM-M13 complex...