Michael Z. Lin - US grants

DESCRIPTION (provided by applicant): The synthesis of proteins by ribosomes is a fundamental step in the expression of genetic information by living organisms. A simple, reliable, rapid, and generalizable method for stopping this fundamental process for a specific protein with a drug would be an enormously useful tool in biomedical and translational research. It would benefit basic studies of protein function by allowing conditional expression of proteins of interest. It would provide a means for regulating protein production from gene and cellular therapies in vivo. A method for controlling synthesis of specific proteins would also allow assessment of how inducible synthesis of specific proteins contributes to particular biological responses or to disease, a line of investigation which has not been possible. In the proposed work, we will develop a novel generalizable method for shutting off synthesis of genetically tagged proteins using a small-molecule drug, and use this method to address outstanding questions on the role of new protein synthesis in nervous system adaptation. We will express proteins of interest as fusions to a potent degradation signal that undergoes autocatalytic removal. By default, the proteins will be released from the degradation signal and function normally. Application of a specific non-toxic cell-permeable drug will preserve the degradation signal on subsequently synthesized proteins, leading to their rapid degradation. We will perform experiments to validate the utility of this method in primary cells and animals, to determine the protein degradation pathways on which this method relies, and to extend the strategy to controlling production of secreted proteins. We will further use this method to test a long-standing hypothesis that synthesis of specific synapse-regulating proteins is required for memory consolidation in transgenic mice. If successful, these experiments will be groundbreaking in establishing a completely new method for controlling protein production that is rapid, robust, simple, and generalizable. The proposed research will have broad benefits in biomedical research by providing a generic method for regulating protein expression that allows more rapid kinetics than transcriptional control methods but, like transcriptional regulation, produces functional proteins without a permanent fusion tag. This work may thus facilitate studies on protein functions in general, including genome-wide screens, and provide a means for tightly controlling gene and cellular therapies in vivo. This work will also produce the first experimental tools capable of addressing the importance of specific protein synthesis events in normal physiology and disease. Thus the proposed experiments have the potential to produce a major advance in our ability to control protein function for elucidating biological mechanisms and for controlling biological therapies.

1134416
Lin

Being able to both control and monitor neuronal activity is critical for learning how neuronal
circuits process information and make decisions. However, while powerful tools to control neuronal firing
using genetically encoded light-activated channels have recently been developed, limitations of current
genetically encoded sensors of neuronal activity severely restrict their use for monitoring brain function.
In particular, calcium sensors are slow and cannot report on subthreshold depolarizations. While voltage
sensors could in principle be used to follow fast trains of action potentials and monitor subthreshold
depolarizations, the low dynamic range of existing sensors makes them unsuitable for most experiments
in vivo. Moreover, there are presently no calcium or voltage sensors that are efficiently excited by red
(>550nm) wavelengths, a region of the spectrum that would enable their concurrent use with blue/greenabsorbing
light-activated channels.
The goal of this proposal is to address these needs by developing a new generation of genetically
encoded voltage biosensors with significantly improved dynamic range and speed and the ability to be
used concurrently with light-activated channels. This work will enable finer and more powerful functional
dissection of the nervous system, while also establishing new protein engineering methods. The expertise
of the lab in fluorescent proteins engineering will be leveraged to educate future scientists and engineers
about research in protein engineering, and to introduce educators to the utility of genetically encoded
biosensors for teaching scientific concepts in laboratory courses.

Intellectual Merit
The engineering objective of this proposal is to apply knowledge of fluorescence spectroscopy
and protein structure to quantitatively improve the performance of an existing class of voltage sensors
(Aim 1) and to develop an entirely new class of voltage sensors (Aim 2). In the first aim, we will increase
dynamic range via rational optimization of Förster resonance energy transfer (FRET) between fluorescent
proteins linked to a voltage sensing domain. In the second aim, we will use a conformationally sensitive
red fluorescent protein to report on movements of a voltage sensing domain with rapid kinetics. For both
aims, we will employ rational design combined with comprehensive saturation mutagenesis and screening
of important sites.
This project will be a pioneering study in molecular engineering in two ways. It will be the first to
rationally apply predictions from modeling of Förster resonance energy transfer to identify the factors
limiting the fluorescence output response, and then to perform protein engineering to comprehensively
address those factors. Second, this project will be the first to combine comprehensive screening with
atomic-level structural knowledge of sensing domains and fluorescent proteins in order to create an
entirely new class of allosteric voltage sensors; this research will thus give insight on the relative
usefulness of prior knowledge and screening to the engineering of genetically encoded biosensors.

Broader Impacts
Voltage sensors developed through this project will be promptly disseminated to the larger
scientific community. These sensors will have a broad impact on the field of neuroscience by enabling
robust voltage sensing and concurrent control and readout of neuronal activity. Our work will also impact
the field of bioengineering by validating concepts and establishing strategies for how to optimize the
design of fluorescent protein-based sensors using energy transfer or allostery.
The proposed project will provide an excellent opportunity to introduce undergraduates and high
school students, including students from disadvantaged backgrounds, to research in molecular
bioengineering. It will also provide the opportunity to introduce educators to the use of genetically
encoded biosensing to create exciting laboratory lessons that teach important basic concepts while
demonstrating the results of recent research in protein engineering.

DESCRIPTION (provided by applicant): Fragile X syndrome is the most common genetic cause of autism, occurring in 1 out of 6000 births. Affected patients also suffer from mental retardation and in some cases seizures. Current treatments involve the use of drugs to ameliorate mood and attention symptoms and to prevent seizures, but are not able to restore normal learning and emotional function. A molecular-level understanding of the neuronal defects in Fragile X syndrome will be necessary for the rational design of therapies to address the underlying cause of the disease. The protein mutated in the disease, the Fragile X mental retardation protein (FMRP), is required for regulating protein synthesis at activated synapses, the communication points between neurons. A large body of evidence suggests that the normal strengthening and weakening of synapses that underlies learning requires the careful regulation of protein synthesis by synaptic activity. Experiments have also suggested a role for FMRP in regulating both synaptic strengthening and weakening. However, the precise relationship between synaptic strengthening and weakening, protein synthesis, and FMRP is poorly understood. For instance, which proteins are synthesized during, utilized in, or required for synaptic strengthening and weakening, and which of these events are affected by FMRP loss, is not known. Research on the function of FMRP in activity-dependent local protein synthesis has been limited by the low sensitivity and resolution of methods for assessing and controlling protein synthesis in living neurons. We have developed new molecular tools that allow the real-time tracking and control of new protein synthesis and the visualization of kinase pathways involved in activity-induced protein synthesis. We propose to use these tools to examine the specificity of protein synthesis responses in synaptic strengthening versus weakening, and to study the effect of FMRP loss on these responses. We will also determine which new proteins are normally required for long-lasting synaptic plasticity, and how FMRP loss might alter those requirements. These studies will provide insight into the regulation and function of synaptic protein synthesis in persistent synaptic plasticity, identify potential molecular targets for therapeutic intervention, and produce new technologies that can benefit the larger neuroscience community.

DESCRIPTION (provided by applicant): Optical control of genetically defined protein activities, i.e. optogenetic regulation of proteins, has long been a dream in biology. If we could develop a generalizable method to optically control activities of proteins of interest, it could profoundly transform biological experimentation and impart new capabilities to gene- and cell-based therapies. For instance, the cellular functions that proteins coordinate, such as survival, apoptosis, differentiation, migration or connectivity (in the nervous system) could be controlled in vivo to study organismal physiology with micron-level resolution. Therapeutically implanted cells could be similarly controlled by light to precisely focus treatment at desired anatomical locations. There are countless other possible applications. Given these advantages of optical protein regulation, considerable efforts have been expended to adapt known light-responsive signaling domains to regulate mammalian proteins. However, existing strategies require extensive screening to create light-responsive proteins, or rely on protein relocalization to indirectly regulate activity. As a result, these methods have been used to control only a few proteins. Thus there exists a need for a method to create light-regulated proteins of interest that is generalizable. We have recently discovered a new class of light-mediated protein-protein interaction, and translated this discovery into a generalizable method for controlling protein activities with light. We hypothesized that fluorescent proteins (FPs) could undergo light-dependent conformational changes that drive changes in oligomerization state. Indeed, we found that tetrameric and dimeric variants of the reversibly photoswitching green FP Dronpa undergo dissociation as they are switched from bright to dark states by cyan light. We then discovered that fusing Dronpa domains at both ends of an enzymatic domain of interest cages it in the dark but allows uncaging upon illumina

? DESCRIPTION (provided by applicant): Attaining effective genetically encoded optical voltage-indicators has been a longstanding goal in neuroscience research and is a key near-term aim of the BRAIN Initiative. Unlike small molecule sensors or hybrids of fluorescent proteins with organic molecules, optical voltage-indicators that can be fully encoded genetically are readily combined with genetic tools and viral delivery methods that enable long-term expression and chronic imaging studies without addition of exogenous agents. Genetically encoded Ca2+-sensors offer similar targeting advantages, but Ca2+-imaging fails to reveal individual spikes in many neuron types, poorly captures sub- threshold membrane dynamics, and has insufficient temporal resolution to capture spike timing to better than ~50-100 ms. Voltage-indicators directly sense the membrane potential and promise faithful reporting of spike waveforms, spike bursts and sub-threshold dynamics, in cells targeted by their genetic class or connectivity. An ideal voltage-indicator would produce large fluorescence responses, to facilitate spike detection, and have millisecond-scale kinetics, to study synchrony and spike-timing aspects of neural coding. However, prior protein voltage-indicators have generally suffered performance-limiting tradeoffs between modest brightness, sluggish kinetics, and limited signaling dynamic range in response to action potentials. To date, no protein voltage-indicator combines the attributes needed for accurate reporting of voltage activity in behaving animals. However, if such a sensor emerged, this would likely have even greater impact on brain science than the surge in research enabled by recent advanced versions of the GCaMP Ca2+-indicator. This proposal seeks to create broad voltage-imaging capabilities and involves two Co-PDs who are highly experienced in fluorescence imaging of neural activity. Working collaboratively, we recently created two new classes of voltage-indicators, of distinct colors and voltage-sensing mechanisms, each of which has substantially superior signaling fidelity than earlier protein voltage-indicators while offering faster kinetics and higher brightness. Using thes two sensor types, we have imaged fast spike trains in cultured neurons and brain slices. Calculations using signal detection theory show our indicators are now on the brink of transitioning into a mainstay approach to monitor large numbers of individual neurons in behaving animals. To enact this, we will use novel massively parallel methods to screen variants of our protein indicators at 100-1000¿ greater throughput than screening methods used previously in the field. We will validate and iteratively optimize the resulting indicators in cultred neurons, mammalian brain slices, and behaving flies, nematodes and mice, by using signal detection theory to benchmark indicator performance. To accompany these voltage-indicators, we will also create imaging instrumentation custom-designed for high-speed (~1 kHz) voltage-imaging in awake head-restrained and freely behaving mice. If our work succeeds, it will be a game-changer for brain research, propelling studies of how cells and circuits function normally and go awry in disease.

Recording the electrical impulses of many individual neurons in intact brain circuits in real time would enable a detailed understanding of how the brain processes information. Technologies for high-fidelity large-scale voltage sensing with cellular resolution would also provide new high-resolution methods for analyzing for how diseases of the brain impact circuit function. However, current methods lack the ability to detect the rich variety of electrical impulses in large numbers of neurons in deep locations in the brain. Imaging of activity-induced calcium transients using genetically encoded calcium indicators and two-photon (2P) microscopy have led to tremendous progress in our understanding of how individual neurons and neuronal populations participate in information representation and circuit plasticity. This now-established paradigm has demonstrated the benefits of combining genetically encoded optical reporters, which allow for investigation of specific neuronal types over time and with minimal perturbation, with 2P microscopy, which enables recording of optical signals in multiple neurons through hundreds of microns of brain tissue. We aim to develop a new paradigm for activity imaging using genetically encoded voltage indicators (GEVIs) and fast 2P imaging. In contrast to calcium indicators, voltage indicators can provide information on subthreshold voltage changes, which form the basis of neuronal computation and modulate excitability, and on timing and order of neuronal action potential firing events, all basic essential information required for understanding information processing in brain circuits. However, in vivo voltage imaging is currently limited by two constraints. First, further improvements to GEVI brightness, responsivity, wavelengths, and localization would ease the detection of electrical events. Second, the speed of conventional 2P imaging is insufficient for large-scale voltage imaging with single-cell resolution. In this project, we will optimize the recording of electrical activity from many individual neurons in the brain at high speed and at depth, integrating the expertise of four groups with expertise in GEVI engineering, fast 2P scanning methods, and systems neuroscience. Our approach combines optimization of the GEVI class that currently responds best to 2P illumination with improvement of fast random-access multi-photon scanning methods that can capture these fast optical signals from large numbers of neurons. Specifically, we will carry out the following aims: (1) Development of ASAP-family GEVIs along axes of brightness, responsivity, wavelength, and subcellular localization, and comprehensive validation in brain tissue under 2P illumination, (2) development of third-generation (3G) RAMP with motion correction and enhanced throughput via light patterning and excitation and emission multiplexing, and (3) recording voltage from hundreds of neurons in vivo with 1ms precision with RAMP microscopy and GEVIs.

ABSTRACT Recording the electrical impulses of individual neurons in intact brain circuits in real time has been a longstanding goal in neuroscience. One potentially widely applicable use of voltage recording would be to test postsynaptic responses upon physiological or optogenetic activation of presynaptic partners. Recording a neuron while its inputs are controlled would enable a detailed understanding of how individual neurons process information. This understanding becomes important when circuity is altered in disease, e.g. in the striatum with Parkinson's or Huntington's diseases, as it would help explain the pathogenesis of the mutant phenotype and suggest possible therapies. However, the only way now to reliably measure membrane voltage in vivo is to perform electrophysiology, whose difficulty and low throughput hamper widespread adoption. We aim to develop a new paradigm for determining the input-output relationshps of neurons using genetically encoded voltage indicators (GEVIs) and two-photon imaging. GEVIs can provide information on subthreshold voltage changes, which form the basis of neuronal computation and modulate excitability, and on timing and order of neuronal action potentials. These constitute basic essential information required for understanding information processing in brain circuits. However, in vivo voltage imaging is currently limited. No published GEVIs respond with sufficient speed and amplitude for spike detection in single trials under two-photon excitation. In this project, we will develop methods to record electrical activity from individual neurons in the brain at depth using two- photon microscopy. Our approach combines engineering of GEVIs that can respond to two-photon illumination with the establishment of conditions for using GEVIs in brain slices and living brains. Specifically, we will carry out the following aims: (1) Generate brighter and more responsive variants of ASAP2s, the best performer under two-photon excitation, and of Ace-mNeonGreen, a leading performer under one-photon excitation; (2) validate GEVI variants for their ability to report contributions of specific inputs to subthreshold and action potential responses in a variety of neurons of the fly visual system, in single trials, in vivo; and (3) systematically test GEVI performance under two-photon excitation in mouse striatal spiny projection neurons in ex vivo acute brain slices and in living mice in vivo, using GEVIs to determine the role of cell type- specific inputs to a recently discovered phenomenon of long-lasting dendritic voltage plateaus. This project will integrate the expertise of three groups spanning protein engineering, optical method development, and systems neuroscience to improve two-photon imaging of GEVIs so they can be used to image voltage transients in single trials. If successful, this project will open up in vivo two-photon imaging of GEVIs to many interested researchers, potentially catalyzing a transformation in how we measure neuronal responses in living brains.

PROJECT SUMMARY Biomolecules with the capacity for diverse molecular recognition are essential reagents in biomedical research and have been recently developed into uniquely efficacious anti-cancer treatments. In particular, antibody mimetic proteins (AMPs) that function both inside and outside cells have been useful for controlling target protein localization, blocking functional interfaces on target proteins, and mediating cytotoxic effects to target-expressing cancer cells. However, a crucial missing feature of existing AMPs is the ability for target binding to be controlled remotely, in real time, and in defined points in space. Optical control of AMP binding would be immensely and widely useful throughout the biosciences by enabling protein sequestration or functional blockade in living cells to be controlled exquisitely in time and space. In particular, photocontrollable AMPs would be powerful reagents for controlling the activity endogenous proteins with spatiotemporal specificity, especially proteins for which no specific small molecule inhibitors exist. Photocontrollable AMPs could also be used to direct immune cells to cancer cells at specific times or at specific locations in the body. We propose to develop the ability to optically control AMP-target interactions using protein domains that can be photoswitched from dimeric to monomeric states by cyan light. We propose that, by inserting two copies of these photoswitchable domains to constant (non-variable) regions in AMPs, we can create AMPs that do not interact with their targets in the dark due to binding site occlusion, but that do bind to targets after cyan illumination. All possible rational designs will be explored, involving using scaffolds of the DARPin and repebody families unmodified or after topological modification, and inserting photodissociable domains at termini and/or interior loops. Specifically, we will (1) engineer photoswitchable DARPins and repebodies by inserting photoswitchable domains into loops and termini of existing DARPins, (2) Engineer photoswitchable DARPins by redesigning the framework to introduce new sites for photodissociable domain insertion while improving protein stability, and (3) Test the ability of photoswitchable DARPins and repebodies to perform light-controlled inhibition, relocalization, or degradation of proteins inside cells and recruitment of immune cells to cells expressing cancer antigens. If successful, this work will establish photoswitchable AMPs as a new type of reagent with exceptionally broad applicability in biology. By allowing binding to nearly any endogenous protein to be remotely control with high spatiotemporal precision, photoswitchable AMPs can give researchers the ability to investigate dynamic processes in living cells in unprecedented detail, and give clinicians the ability to recruit the immune system to specific targets at specific times and in specific regions of the body.

PROJECT SUMMARY Small numbers of neurons can play outsized roles in brain function. For example, neuromodulatory circuits that contain only thousands of neurons project throughout the brain to control the activity of billions of neurons in other brain regions. These neuromodulatory circuits, which secrete the neuromodulators dopamine, serotonin, norepineprhine, acetylcholine, and histamine, regulate essential brain functions such as learning, eating, mating, and socialization. Importantly, alteration of neuromodulatory circuit function underlies addiction, e.g. dopaminergic, serotonergic, and noradrenergic functions are potentiated by cocaine and amphetamines, and cholinergic functions by nicotine. However, very little work has addressed how neuromodulatory circuits respond in real time to environmental stimuli, behavioral output, or drug use. In addition, little is known about how these neuromodulatory circuits develop as animals mature. In the cortex and hippocampus, genetically encoded calcium indicators (GECIs) and two-photon microscopy together have been enormously successful in relating activities of specific types of neurons to various stimuli or behavioral outputs. In contrast, efforts to apply calcium imaging to neuromodulatory circuits have proceeded on a far smaller scale, mostly due to their difficult anatomy. The deep locations of neuromodulatory nuclei near other vital brain regions make the placement of optical lenses or fibers very difficult to perform without creating severe behavioral or functional deficits. Thus, what is needed, and what does not exist, is a way to non- invasively record the total the activity of a specific neuromodulatory network from outside the animal. With the long-term goal of enabling noninvasive activity recording of specific neuronal circuits in living animals, we propose to create and implement GECIs that operate not via fluorescence but via bioluminescence, in which a luciferase enzyme reacts with a chemical substrate to produce light in a calcium-dependent manner. The lack of endogenous luciferases leads to background that is negligible, so high contrast can be achieved even with external detectors. In preliminary work, we have generated a calcium-dependent variant that demonstrates 7-fold increase in photonic output in response to calcium, a similar response ratio to the widely used fluorescent reporter GCaMP3. Compared to other bioluminescent calcium reporters, this reporter produces orders of magnitude more photons above 600 nm, to which tissue is relatively transparent. This reporter, which we name red calcium- modulated bioluminescent indicator (orange CaMBI), has been used successfully in cultured neurons. We now propose to develop methods to non-invasively visualize the activity of specific neuromodulatory circuits in freely behaving mice, by (1) testing the ability of CaMBI expressed throughout the brain and substrates injected intravenously, intraperitoneally, intraventricularly to produce luminescence from the brain in an activity- dependent manner, (2) making a transcranial head-mounted optical recording device, and test the ability to detect activity in neuromodulatory circuits, and (3) futher improving CaMBI for brightness and calcium responsiveness.

ABSTRACT Although acetylcholine (ACh)-secreting neurons only constitute a small fraction of the total neurons in an animal brain, they play a critical role in regulating essential brain functions. In particular, the dysregulation of cholinergic neurons has been connected to the neurological deficits observed in Alzheimer?s disease (AD), the most common neurological disorder of aging. However, while mouse models recapitulating the features of AD have been established, the development of effective therapies or preventions has been hampered by our ability to longitudinally monitor cholinergic function and correlate it to behavioral changes during disease development. In recent years, the development of genetically encoded fluorescent indicators for neurotransmitters has made enormous progress in real-time imaging neurotransmitter release in live mouse, thereby enabling many exciting discoveries relating the activity of specific neurotransmitters to various behavioral outputs. However, in vivo fluorescent imaging in the brain suffers from the need to invasively insert illumination and recording devices, which could easily create behavioral or functional deficits, especially when the region of interest is located deeply. Thus, what is needed, and what does not exist, is a way to non-invasively and longitudinally observe the release of neurotransmitters such as ACh from outside the animal. We propose to create and validate ACh indicators that operate not via fluorescence but via bioluminescence, in which a luciferase enzyme oxidizes a chemical substrate to produce light in a neurotransmitter-dependent manner. No external excitation light is needed for bioluminescent imaging, and auto-bioluminescence is usually missing in mammals, therefore sensitive imaging in deep tissues can be easily achieved with external detectors. In our preliminary work, we demonstrated that Antares, a highly catalytic and red-emitting luciferase we engineered, when coupled with novel substrates that we developed, was able to produce 55-fold brighter bioluminescence in mouse brain, compared to the commonly used firefly luciferase. This state-of-the-art luciferase-luciferin pair now opens the door to create sensitive bioluminescent reporters that function in the brain. We now propose to create neurotransmitter bioluminescent indicators (NeuBIs), starting with ACh as the primary target. Specifically, the protein-based ACh indicator will be developed from the luciferase Antares, and either (1) a bacterial ACh binding domain, or (2) muscarinic receptors. The created ACh indicators will be tested in cultured neurons and mice to image ACh release. Once established, we envision these ACh indicators will be widely used for non-invasive recording of cholinergic activity in mouse models of AD, and can serve as templates for the engineering of other neurotransmitter bioluminescent indicators.

The broader impact of this I-Corps program is the development of bioluminescence tools that may be applied to drug discovery and optimization, particularly for the non-invasive imaging of animals. To date, bioluminescence imaging in the pharmaceutical industry has been limited due to poor light penetrance and enzyme instability. Fundamental research has contributed to the advancement of bioluminescence imaging. The goal is to bridge the gap and expand the use of bioluminescent reporters. Specifically, the proposed technology will use biosensors for rapidly evaluating drug candidates in laboratory animals, in addition to cell cultured cells. This technology may have a significant impact on central nervous system (CNS) drug development and accelerate the optimization of drugs for brain malignancies. This tool may lead to improved compounds with better blood brain barrier (BBB) penetrance and better activity in the brain, while also preventing the testing of suboptimal compounds in human volunteers and patients.

This I-Corps project is based on the development of new drug screening platforms that enable non-invasive detection of drug activity and efficacy in living animals. Recently, bioluminescent reporters have been engineered to be brighter and red-shifted, thereby allowing deep tissue imaging of biological events. These molecular tools enable the detection of drug activity in an approach that is label-free, non-invasive, and pathway-specific. In addition, these biosensors may be used to monitor drug activity in living animals, thus circumventing the numerous limitations of terminal analyses. By evaluating targeted drug candidates in vivo, without termination, the performance and efficacy of the compounds can be more accurately appraised. This is especially valuable for optimizing molecules penetrating the central nervous system (CNS), in which the blood-brain barrier (BBB) has presented significant challenges in CNS drug development. The goal of this project is to determine the ways in which these biosensors could facilitate the rapid assessment of CNS compounds, and when these tools may have the most impact in the drug development pipeline.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

ABSTRACT To understand how the brain functions in health, and how sensory, motor, and cognitive functions are affected in disease, it is crucial to be able to record the activities of large numbers of individual neurons in real time. In the past two decades, calcium imaging in neuronal cell bodies has provided a conveniently qualitative view of neuronal activity, allowing action potential firing in specific neuron types or in various brain regions to be correlated with sensory input, decision-making, or internal representations of emotional or physiological parameters. However, somatic calcium is generally insensitive to subthreshold activity, i.e. to synaptic inputs that depolarize the membrane potential without eliciting action potentials, and lacks the temporal precision to determine the timing relationship between action potentials within a circuit. Our understanding of the brain would beneift greatly from understanding how neuronal circuits use transmembrane voltage to represent and process information. Outstanding questions include how neuronal types differ in their summatation of inputs to initiate an action potential output, how neuronal circuits extract salient features or makes a decision based on complex patterns of input activity, and how experience or neuromodulation or disease affects these processes. We propose to address this problem by creating a class of high-performance genetically encoded voltage indicators (GEVIs) to record both subthreshold and spiking activity in large numbers of neurons in living animals. In particular, we find that positively tuned GEVIs have the potential for detecting spikes with many times greater signal-to-noise ratio than GECIs while achieving useful discriminability of subthreshold potentials. We propose an intense effort to develop such positively tuned GEVIs toward ideal performance specifications identified by quantitative modeling. Aims include (1) comprehensive screening of residues in a prototype positively tuned GEVI to identify positions modulating voltage tuning and fluorescence responsiveness, followed by deep combinatorial mutagenesis of identified sites, (2) validation of indicators in vivo in 1-photon and 2-photon imaging in flies and mice, and (3) development of an ultra-high-throughput single-cell screening system to further accelerate GEVI improvement.