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.

Correction to: A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

In the online version of the article [ 1 ], Figure S1 was mistakenly replaced with Figure 1.

A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

BACKGROUND: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores. RESULTS: Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo. CONCLUSION: K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes.

The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These 'Twitch' sensors are based on the C-terminal domain of Opsanus troponin C. Their FRET responses were optimized by a large-scale functional screen in bacterial colonies, refined by a secondary screen in rat hippocampal neuron cultures. We tested the in vivo performance of the most sensitive variants in the brain and lymph nodes of mice. The sensitivity of the Twitch sensors matched that of synthetic calcium dyes and allowed visualization of tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes. Given their ratiometric readout, their brightness, large dynamic range and linear response properties, Twitch sensors represent versatile tools for neuroscience and immunology.

Line temporal focusing characteristics in transparent and scattering media.

Line illumination geometries have advantageous properties for temporal focusing nonlinear microscopy. The characteristics of line temporal focusing (LITEF) in transparent and scattering media are studied here both experimentally and using numerical model simulations. We introduce an approximate analytical formula for the dependence of axial sectioning on the laser and microscope's parameters. Furthermore, we show that LITEF is more robust to tissue scattering than wide-field temporal focusing, and can penetrate much deeper into scattering tissue while maintaining good sectioning capabilities. Based on these observations, we propose a new design for LITEF-based tissue imaging at depths that could potentially exceed the out-of-focus physical excitation limit.

Large-scale recording of neuronal activity in freely-moving mice at cellular resolution.

Current methods for recording large-scale neuronal activity from behaving mice at single-cell resolution require either fixing the mouse head under a microscope or attachment of a recording device to the animal's skull. Both of these options significantly affect the animal behavior and hence also the recorded brain activity patterns. Here, we introduce a different method to acquire snapshots of single-cell cortical activity maps from freely-moving mice using a calcium sensor called CaMPARI. CaMPARI has a unique property of irreversibly changing its color from green to red inside active neurons when illuminated with 400 nm light. We capitalize on this property to demonstrate cortex-wide activity recording without any head fixation, tethering, or attachment of a miniaturized device to the mouse's head. Multiple cortical regions were recorded while the mouse was performing a battery of behavioral and cognitive tests. We identified task-dependent activity patterns across motor and somatosensory cortices, with significant differences across sub-regions of the motor cortex and correlations across several activity patterns and task parameters. This CaMPARI-based recording method expands the capabilities of recording neuronal activity from freely-moving and behaving mice under minimally-restrictive experimental conditions and provides large-scale volumetric data that are currently not accessible otherwise.

Longitudinal in vivo monitoring of axonal degeneration after brain injury.

Traumatic brain injury (TBI)-induced axonal degeneration leads to acute and chronic neuropsychiatric impairment, neuronal death, and accelerated neurodegenerative diseases of aging, including Alzheimer's and Parkinson's diseases. In laboratory models, axonal degeneration is traditionally studied through comprehensive postmortem histological evaluation of axonal integrity at multiple time points. This requires large numbers of animals to power for statistical significance. Here, we developed a method to longitudinally monitor axonal functional activity before and after injury in vivo in the same animal over an extended period. Specifically, after expressing an axonal-targeting genetically encoded calcium indicator in the mouse dorsolateral geniculate nucleus, we recorded axonal activity patterns in the visual cortex in response to visual stimulation. In vivo aberrant axonal activity patterns after TBI were detectable from 3 days after injury and persisted chronically. This method generates longitudinal same-animal data that substantially reduces the number of required animals for preclinical studies of axonal degeneration.

Remotely scanned multiphoton temporal focusing by axial grism scanning.

A simple technique for remote scanning of the focal plane in temporal focusing multiphoton microscopy is demonstrated both theoretically and experimentally. A new on-axis light propagation optical setup design enables this scanning, which was considered not feasible in previous studies. The focal plane is axially displaced by the movement of a remote optical device, consisting of a double prism grating, and optionally a cylindrical lens. The displacement is linear, and its slope is inversely proportional to the square of the optical system's magnification.

Enhanced detection sensitivity of neuronal activity patterns using CaMPARI1 vs. CaMPARI2.

Calcium-modulated photoactivatable ratiometric integrator (CaMPARI) is a calcium ion (Ca2+)- and light-dependent genetically encoded fluorescent activity integrator that can capture snapshots of neuronal activity through an irreversible process known as photoconversion. This unique property was previously used to label neurons based upon their tuning properties in order to map synaptic connectivity and to record large-scale neuronal activity in freely moving mice without attaching any mechanical device to them. The latest version of CaMPARI (CaMPARI2) was engineered to enhance the contrast generated by photoconverting the green protein to the activity-dependent red form and to reduce the Ca2+-independent photoconversion rate compared to the first generation of CaMPARI (CaMPARI1). However, here we show that this optimization process also resulted in reduced photoconversion efficiency of active neurons in the mouse cortex and hippocampus. Through side-by-side comparison of the two CaMPARI sensors under several experimental conditions, we show that CaMPARI1 exhibits a substantially higher red-to-green ratio in active cells than CaMPARI2. In addition, we show that CaMPARI1 also functions as a more sensitive traditional Ca2+ sensor than CaMPARI2 by producing larger activity-driven dynamic fluorescence changes in the observed neurons. Therefore, we conclude that during the optimization process of CaMPARI2, some of the sensor's characteristics were not predicted properly by in vitro screening assays, and therefore in vivo screening and validation steps should be included in future optimization attempts to increase the predictability of screening pipelines.

Numerical evaluation of temporal focusing characteristics in transparent and scattering media.

Temporal focusing is a simple approach for achieving tight, optically sectioned excitation in nonlinear microscopy and multiphoton photo-manipulation. Key applications and advantages of temporal focusing involve propagation through scattering media, but the progressive broadening of the temporal focus has not been characterized. By combining a detailed geometrical optics model with Monte-Carlo scattering simulations we introduce and validate a simulation strategy for predicting temporal focusing characteristics in scattering and non-scattering media. The broadening of the temporal focus width with increasing depth in brain tissue is studied using both simulations and experiments for several key optical geometries, and an analytical approximation is found for the dependence of this broadening on the microscope's parameters in a transparent medium. Our results indicate that a multiphoton temporal focus has radically different broadening characteristics in deep tissue than those of a spatial focus.

Cellular-resolution monitoring of ischemic stroke pathologies in the rat cortex.

Stroke is a leading cause of disability in the Western world. Current post-stroke rehabilitation treatments are only effective in approximately half of the patients. Therefore, there is a pressing clinical need for developing new rehabilitation approaches for enhancing the recovery process, which requires the use of appropriate animal models. Here, we demonstrate the use of nonlinear microscopy of calcium sensors in the rat brain to study the effects of ischemic stroke injury on cortical activity patterns. We longitudinally recorded from thousands of neurons labeled with a genetically-encoded calcium indicator before and after an ischemic stroke injury in the primary motor cortex. We show that this injury has an effect on the activity patterns of neurons not only in the motor and somatosensory cortices, but also in the more distant visual cortex, and that these changes include modified firing rates and kinetics of neuronal activity patterns in response to a sensory stimulus. Changes in neuronal population activity provided animal-specific, circuit-level information on the post-stroke cortical reorganization process, which may be essential for evaluating the efficacy of new approaches for enhancing the recovery process.

Reversible Loss of Hippocampal Function in a Mouse Model of Demyelination/Remyelination.

Demyelination of axons in the central nervous system (CNS) is a hallmark of multiple sclerosis (MS) and other demyelinating diseases. Cycles of demyelination, followed by remyelination, appear in the majority of MS patients and are associated with the onset and quiescence of disease-related symptoms, respectively. Previous studies in human patients and animal models have shown that vast demyelination is accompanied by wide-scale changes to brain activity, but details of this process are poorly understood. We used electrophysiological recordings and non-linear fluorescence imaging from genetically encoded calcium indicators to monitor the activity of hippocampal neurons during demyelination and remyelination over a period of 100 days. We found that synaptic transmission in CA1 neurons was diminished in vitro, and that neuronal firing rates in CA1 and the dentate gyrus (DG) were substantially reduced during demyelination in vivo, which partially recovered after a short remyelination period. This new approach allows monitoring how changes in synaptic transmission induced by cuprizone diet affect neuronal activity, and it can potentially be used to study the effects of therapeutic interventions in protecting the functionality of CNS neurons.

Thy1 transgenic mice expressing the red fluorescent calcium indicator jRGECO1a for neuronal population imaging in vivo.

Calcium imaging is commonly used to measure the neural activity of large groups of neurons in mice. Genetically encoded calcium indicators (GECIs) can be delivered for this purpose using non-invasive genetic methods. Compared to viral gene transfer, transgenic targeting of GECIs provides stable long-term expression and obviates the need for invasive viral injections. Transgenic mice expressing the green GECI GCaMP6 are already widely used. Here we present the generation and characterization of transgenic mice expressing the sensitive red GECI jRGECO1a, driven by the Thy1 promoter. Four transgenic lines with different expression patterns showed sufficiently high expression for cellular in vivo imaging. We used two-photon microscopy to characterize visual responses of individual neurons in the visual cortex in vivo. The signal-to-noise ratio in transgenic mice was comparable to, or better than, mice transduced with adeno-associated virus. In addition, we show that Thy1-jRGECO1a transgenic mice are useful for transcranial population imaging and functional mapping using widefield fluorescence microscopy. We also demonstrate imaging of visual responses in retinal ganglion cells in vitro. Thy1-jRGECO1a transgenic mice are therefore a useful addition to the toolbox for imaging activity in intact neural networks.

Improved methods for marking active neuron populations.

Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue.

Neural signatures of dynamic stimulus selection in Drosophila.

Many animals orient using visual cues, but how a single cue is selected from among many is poorly understood. Here we show that Drosophila ring neurons-central brain neurons implicated in navigation-display visual stimulus selection. Using in vivo two-color two-photon imaging with genetically encoded calcium indicators, we demonstrate that individual ring neurons inherit simple-cell-like receptive fields from their upstream partners. Stimuli in the contralateral visual field suppressed responses to ipsilateral stimuli in both populations. Suppression strength depended on when and where the contralateral stimulus was presented, an effect stronger in ring neurons than in their upstream inputs. This history-dependent effect on the temporal structure of visual responses, which was well modeled by a simple biphasic filter, may determine how visual references are selected for the fly's internal compass. Our approach highlights how two-color calcium imaging can help identify and localize the origins of sensory transformations across synaptically connected neural populations.

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.

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.

All-optical bidirectional neural interfacing using hybrid multiphoton holographic optogenetic stimulation.

Our understanding of neural information processing could potentially be advanced by combining flexible three-dimensional (3-D) neuroimaging and stimulation. Recent developments in optogenetics suggest that neurophotonic approaches are in principle highly suited for noncontact stimulation of network activity patterns. In particular, two-photon holographic optical neural stimulation (2P-HONS) has emerged as a leading approach for multisite 3-D excitation, and combining it with temporal focusing (TF) further enables axially confined yet spatially extended light patterns. Here, we study key steps toward bidirectional cell-targeted 3-D interfacing by introducing and testing a hybrid new 2P-TF-HONS stimulation path for accurate parallel optogenetic excitation into a recently developed hybrid multiphoton 3-D imaging system. The system is shown to allow targeted all-optical probing of in vitro cortical networks expressing channelrhodopsin-2 using a regeneratively amplified femtosecond laser source tuned to 905 nm. These developments further advance a prospective new tool for studying and achieving distributed control over 3-D neuronal circuits both in vitro and in vivo.