Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials (APs) in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by several-fold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2–3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.

Photochemical release (uncaging) of bioactive messengers with three-dimensional spatial resolution in light-scattering media would be greatly facilitated if the photolysis could be powered by pairs of IR photons rather than the customary single UV photons. The quadratic dependence on light intensity would confine the photolysis to the focus point of the laser, and the longer wavelengths would be much less affected by scattering. However, previous caged messengers have had very small cross sections for two-photon excitation in the IR region. We now show that brominated 7-hydroxycoumarin-4-ylmethyl esters and carbamates efficiently release carboxylates and amines on photolysis, with one-and two-photon cross sections up to one or two orders of magnitude better than previously available. These advantages are demonstrated on neurons in brain slices from rat cortex and hippocampus excited by glutamate uncaged from N-(6-bromo-7-hydroxycoumarin-4-ylmethoxycarbonyl)-L-glutamate (Bhc-glu). Conventional UV photolysis of Bhc-glu requires less than one-fifth the intensities needed by one of the best previous caged glutamates, ␥-(␣-carboxy-2-nitrobenzyl)-L-glutamate (CNB-glu). Two-photon photolysis with rasterscanned femtosecond IR pulses gives the first threedimensionally resolved maps of the glutamate sensitivity of neurons in intact slices. Bhc-glu and analogs should allow more efficient and three-dimensionally localized uncaging and photocleavage, not only in cell biology and neurobiology but also in many technological applications.

articlesCentral to understanding learning mechanisms at a synaptic level is the idea that lasting functional change can be driven by the coincidence of multiple signals at a single synaptic site 1 . One candidate for such a change is long-term depression of the parallel fiber input to cerebellar Purkinje cells, a form of synaptic plasticity that is thought to underlie several forms of associative motor learning 2 . LTD is induced by coincident activation of PF and CF synaptic inputs, and can last from hours to days 2,3 .Although LTD has been extensively studied, the identity of the coincidence detection mechanism(s) triggered by conjunctive activation of PF and CF synapses is still controversial 4 . One second messenger, cytosolic calcium (Ca 2+ ), is both necessary 5 and sufficient 6 (L. Khiroug, G. Ellis-Davies & G. J. Augustine, Soc. Neurosci. Abstr. 25, 397.3, 1999) to induce a depression of synaptic strength. This indicates that Ca 2+ is a critical link in the induction of LTD. However, in contrast to most other synapses that show Ca 2+ -dependent associative plasticity, PF synapses lack postsynaptic NMDA receptors, which allow Ca 2+ to enter neurons in response to the simultaneous presence of glutamate and depolarization (for review, see ref. 1).Purkinje cells do, however, possess two other molecular mechanisms that could detect conjunctive PF and CF activation to generate a Ca 2+ signal. The first of these is the inositol-1,4, 5-trisphosphate (IP 3 ) receptor 7 , which is synergistically coactivated 8 by IP 3 and Ca 2+ . The IP 3 receptor controls Ca 2+ release from the endoplasmic reticulum and is present in high concentrations in both spines and shafts of Purkinje cell dendrites 9 . IP 3 could be formed via the activation of metabotropic glutamate receptors (mGluR) by glutamate released from PF terminals 10-12 . The source of Ca 2+ could be voltage-gated Ca 2+ channels, which are known to be located on PF spines 13 , and may even be colocalized with the IP 3 receptor on a molecular scale 14 . The depolarization necessary for activation of these Ca 2+ channels could be provided by the complex spike that is triggered by CF input 15 . This mechanism, therefore, predicts that the combination of CF and PF inputs should generate a supralinear Ca 2+ signal in PF spines 7 .A second potential coincidence detection mechanism uses the voltage-gated Ca 2+ channels in PF spines 13 . Because the channel activation curve is highly nonlinear, even a small local depolarization resulting from activation of AMPA channels at the PF synapse 16 could combine with the depolarization by the complex spike to cause supralinear Ca 2+ influx.The involvement of release through IP 3 receptors in the LTD induction pathway is supported by experiments demonstrating that blockers of Ca 2+ release inhibit LTD 6 , and that IP 3 uncaging can cause a form of LTD 6,11 . The metabotropic pathway is implicated because LTD induction can be prevented by mGluR antagonists 17 (but see also ref. 4), and is impaired in a genetic knockout of mGluR1 ...