jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths.

Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.

Bright and photostable chemigenetic indicators for extended in vivo voltage imaging.

Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

High-performance calcium sensors for imaging activity in neuronal populations and microcompartments.

Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.

Photoactivatable drugs for nicotinic optopharmacology.

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

A general method to fine-tune fluorophores for live-cell and in vivo imaging.

Pushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. We refined and extended this strategy, finding that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties of rhodamine dyes with unprecedented precision. This strategy allowed us to establish principles for fine-tuning the properties of fluorophores and to develop a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red. Our results demonstrate the versatility of these new dyes in cells, tissues and animals.

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo.

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

Sensitive red protein calcium indicators for imaging neural activity.

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.

Cortex commands the performance of skilled movement.

Mammalian cerebral cortex is accepted as being critical for voluntary motor control, but what functions depend on cortex is still unclear. Here we used rapid, reversible optogenetic inhibition to test the role of cortex during a head-fixed task in which mice reach, grab, and eat a food pellet. Sudden cortical inhibition blocked initiation or froze execution of this skilled prehension behavior, but left untrained forelimb movements unaffected. Unexpectedly, kinematically normal prehension occurred immediately after cortical inhibition, even during rest periods lacking cue and pellet. This 'rebound' prehension was only evoked in trained and food-deprived animals, suggesting that a motivation-gated motor engram sufficient to evoke prehension is activated at inhibition's end. These results demonstrate the necessity and sufficiency of cortical activity for enacting a learned skill.

Conjunctive input processing drives feature selectivity in hippocampal CA1 neurons.

Feature-selective firing allows networks to produce representations of the external and internal environments. Despite its importance, the mechanisms generating neuronal feature selectivity are incompletely understood. In many cortical microcircuits the integration of two functionally distinct inputs occurs nonlinearly through generation of active dendritic signals that drive burst firing and robust plasticity. To examine the role of this processing in feature selectivity, we recorded CA1 pyramidal neuron membrane potential and local field potential in mice running on a linear treadmill. We found that dendritic plateau potentials were produced by an interaction between properly timed input from entorhinal cortex and hippocampal CA3. These conjunctive signals positively modulated the firing of previously established place fields and rapidly induced new place field formation to produce feature selectivity in CA1 that is a function of both entorhinal cortex and CA3 input. Such selectivity could allow mixed network level representations that support context-dependent spatial maps.