Population signals from neuronal ensembles in cortex during behavior are commonly measured with EEG, local field potential (LFP), and voltage-sensitive dyes. A genetically encoded voltage indicator would be useful for detection of such signals in specific cell types. Here we describe how this goal can be achieved with Butterfly, a voltage-sensitive fluorescent protein (VSFP) with a subthreshold detection range and enhancements designed for voltage imaging from single neurons to brain in vivo. VSFP-Butterfly showed reliable membrane targeting, maximum response gain around standard neuronal resting membrane potential, fast kinetics for single-cell synaptic responses, and a high signal-to-noise ratio. Butterfly reports excitatory postsynaptic potentials (EPSPs) in cortical neurons, whisker-evoked responses in barrel cortex, 25-Hz gamma oscillations in hippocampal slices, and 2- to 12-Hz slow waves during brain state modulation in vivo. Our findings demonstrate that cell class-specific voltage imaging is practical with VSFP-Butterfly, and expand the genetic toolbox for the detection of neuronal population dynamics.

UV-sensitive syndrome (UV s S) is a rare autosomal recessive disorder characterized by photosensitivity and mild freckling but without neurological abnormalities or skin tumors. UV s S cells show UV hypersensitivity and defective transcription-coupled DNA repair of UV damage. It was suggested that UV s S does not belong to any complementation groups of known photosensitive disorders such as xeroderma pigmentosum and Cockayne syndrome (CS). To identify the gene responsible for UV s S, we performed a microcellmediated chromosome transfer based on the functional complementation of UV hypersensitivity. We found that one of the UV s S cell lines, UV s 1KO, acquired UV resistance when human chromosome 10 was transferred. Because the gene responsible for CS group B (CSB), which involves neurological abnormalities and photosensitivity as well as a defect in transcription-coupled DNA repair of UV damage, is located on chromosome 10, we sequenced the CSB gene from UV s 1KO and detected a homozygous null mutation. Our results indicate that previous complementation analysis of UV s 1KO was erroneous. This finding was surprising because a null mutation of the CSB gene would be expected to result in CS features such as severe developmental and neurological abnormalities. On the other hand, no mutation in the CSB cDNA and a normal amount of CSB protein was detected in Kps3, a UV s S cell line obtained from an unrelated patient, indicating genetic heterogeneity in UV s S. Possible explanations for the discrepancy in the genotype-phenotype relationship in UV s 1KO are presented. N ucleotide excision repair (NER) is a versatile DNA repair system that removes a wide range of DNA lesions including UV-induced lesions (1). There are two subpathways in NER. One is transcription-coupled DNA repair (TCR), which preferentially removes DNA damage that blocks ongoing transcription in the transcribed DNA strand of active genes, and the other is global genome repair (GGR), which removes lesions throughout the genome including those from the nontranscribed strand in the active gene (2). There are several NER-deficient disorders, such as xeroderma pigmentosum (XP), Cockayne syndrome (CS), cerebro-oculo-facio-skeletal syndrome, trichothiodystrophy, and UV-sensitive syndrome (UV s S). Hypersensitivity to sunlight is a common hallmark of these rare, autosomal recessive diseases that otherwise are highly heterogeneous with respect to additional features and genetic background. XP patients exhibit a Ͼ1,000-fold increased incidence of sun-induced skin cancer and often experience accelerated neurodegeneration. XP is classified into seven genetic complementation groups (XP-A to XP-G) and a variant form (XP-V) (1). XP-A to XP-G have a defect in TCR and GGR, except for XP-C and XP-E (2), which have a defect in GGR only. XP-V has a normal NER but a defect in translesion DNA synthesis (3). CS is characterized by an abnormality in physical and neurological development with dysmyelination but no predisposition to skin cancer (4). CS is classified into two g...

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Over the last decade, optical neuroimaging methods have been enriched by engineered biosensors derived from fluorescent protein (FP) reporters fused to protein detectors that convert physiological signals into changes of intrinsic FP fluorescence. These FP-based indicators are genetically encoded, and hence targetable to specific cell populations within networks of heterologous cell types. Among this class of biosensors, the development of optical probes for membrane potential is both highly desirable and challenging. A suitable FP voltage sensor would indeed be a valuable tool for monitoring the activity of thousands of individual neurons simultaneously in a non-invasive manner. Previous prototypic genetically-encoded FP voltage indicators achieved a proof of principle but also highlighted several difficulties such as poor cell surface targeting and slow kinetics. Recently, we developed a new series of FRET-based Voltage-Sensitive Fluorescent Proteins (VSFPs), referred to as VSFP2s, with efficient targeting to the plasma membrane and high responsiveness to membrane potential signaling in excitable cells. In addition to these FRET-based voltage sensors, we also generated a third series of probes consisting of single FPs with response kinetics suitable for the optical imaging of fast neuronal signals. These newly available genetically-encoded reporters for membrane potential will be instrumental for future experimental approaches directed toward the understanding of neuronal network dynamics and information processing in the brain. Here, we review the development and current status of these novel fluorescent probes.