Hiroki Mutoh

Imaging brain electric signals with genetically targeted voltage-sensitive fluorescent proteins

Cortical information processing relies on synaptic interactions between diverse classes of neurons with distinct electrophysiological and connection properties. Uncovering the operational principles of these elaborate circuits requires the probing of electrical activity from selected populations of defined neurons. Here we show that genetically encoded voltage-sensitive fluorescent proteins (VSFPs) provide an optical voltage report from targeted neurons in culture, acute brain slices and living mice. By expressing VSFPs in pyramidal cells of mouse somatosensory cortex, we also demonstrate that these probes can report cortical electrical responses to single sensory stimuli in vivo. These protein-based voltage probes will facilitate the analysis of cortical circuits in genetically defined cell populations and are hence a valuable addition to the optogenetic toolbox.

Engineering and characterization of an enhanced fluorescent protein voltage sensor.

Background Fluorescent proteins have been used to generate a variety of biosensors to optically monitor biological phenomena in living cells. Among this class of genetically encoded biosensors, reporters for membrane potential have been a particular challenge. The use of presently known voltage sensor proteins is limited by incorrect subcellular localization and small or absent voltage responses in mammalian cells. Results Here we report on a fluorescent protein voltage sensor with superior targeting to the mammalian plasma membrane and high responsiveness to membrane potential signaling in excitable cells. Conclusions and Significance This biosensor, which we termed VSFP2.1, is likely to lead to new methods of monitoring electrically active cells with cell type specificity, non-invasively and in large numbers, simultaneously.

Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein

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.

Engineering of a Genetically Encodable Fluorescent Voltage Sensor Exploiting Fast Ci-VSP Voltage-Sensing Movements

Ci-VSP contains a voltage-sensing domain (VSD) homologous to that of voltage-gated potassium channels. Using charge displacement (‘gating’ current) measurements we show that voltage-sensing movements of this VSD can occur within 1 ms in mammalian membranes. Our analysis lead to development of a genetically encodable fluorescent protein voltage sensor (VSFP) in which the fast, voltage-dependent conformational changes of the Ci-VSP voltage sensor are transduced to similarly fast fluorescence read-outs.

Activation of cerebellar parallel fibers monitored in transgenic mice expressing a fluorescent Ca2+ indicator protein

Genetically encoded fluorescent Ca2+ indicator proteins (FCIPs) are promising tools to study Ca2+ signaling in large assemblies of nerve cells. Currently, there are few examples of stable transgenic mouse lines that functionally express such sensors in well‐defined neuronal cell populations. Here we report the generation and characterization of transgenic mice expressing an FCIP under the 5′ regulatory sequences of the Kv3.1 potassium channel promoter. In the cerebellar cortex, expression was restricted to granule cells. We first demonstrated reliable measurements of Ca2+ transients from beams of parallel fibers and compared the FCIP signals with intrinsic autofluorescence signals. We demonstrate that, in a transgenic line that exhibits a high expression level of the FCIP, autofluorescence signals are negligible and stimulation‐induced fluorescence transients represent FCIP signals. Using frontal cerebellar slices we imaged antidromic activation of granule cells following electrical stimulation of parallel fibers and orthodromic activation of beams of parallel fibers following electrical stimulation of granule cells. We found that paired pulse‐induced presynaptic Ca2+ transients of parallel fibers are not affected by blockade of N‐methyl‐d‐aspartate receptors.

Genetically encoded fluorescent sensors of membrane potential

Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments.

Second and Third Generation Voltage-Sensitive Fluorescent Proteins for Monitoring Membrane Potential

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.

Red-Shifted Voltage-Sensitive Fluorescent Proteins

Electrical signals generated by nerve cells provide the basis of brain function. Whereas single or small numbers of cells are easily accessible using microelectrode recording techniques, less invasive optogenetic methods with spectral properties optimized for in vivo imaging are required for elucidating the operation mechanisms of neuronal circuits composed of large numbers of neurons originating from heterogeneous populations. To this end, we generated and characterized a series of genetically encoded voltage-sensitive fluorescent proteins by molecular fusion of the voltage-sensing domain of Ci-VSP (Ciona intestinalis voltage sensor-containing phosphatase) to red-shifted fluorescent protein operands. We show how these indicator proteins convert voltage-dependent structural rearrangements into a modulation of fluorescence output and demonstrate their applicability for optical recording of individual or simultaneous electrical signals in cultured hippocampal neurons at single-cell resolution without temporal averaging.

Optical Imaging of Postsynaptic Odor Representation in the Glomerular Layer of the Mouse Olfactory Bulb

Olfactory glomeruli are the loci where the first odor-representation map emerges. The glomerular layer comprises exquisite local synaptic circuits for the processing of olfactory coding patterns immediately after their emergence. To understand how an odor map is transferred from afferent terminals to postsynaptic dendrites, it is essential to directly monitor the odor-evoked glomerular postsynaptic activity patterns. Here we report the use of a transgenic mouse expressing a Ca(2+)-sensitive green fluorescence protein (GCaMP2) under a Kv3.1 potassium-channel promoter. Immunostaining revealed that GCaMP2 was specifically expressed in mitral and tufted cells and a subpopulation of juxtaglomerular cells but not in olfactory nerve terminals. Both in vitro and in vivo imaging combined with glutamate receptor pharmacology confirmed that odor maps reported by GCaMP2 were of a postsynaptic origin. These mice thus provided an unprecedented opportunity to analyze the spatial activity pattern reflecting purely postsynaptic olfactory codes. The odor-evoked GCaMP2 signal had both focal and diffuse spatial components. The focalized hot spots corresponded to individually activated glomeruli. In GCaMP2-reported postsynaptic odor maps, different odorants activated distinct but overlapping sets of glomeruli. Increasing odor concentration increased both individual glomerular response amplitude and the total number of activated glomeruli. Furthermore, the GCaMP2 response displayed a fast time course that enabled us to analyze the temporal dynamics of odor maps over consecutive sniff cycles. In summary, with cell-specific targeting of a genetically encoded Ca(2+) indicator, we have successfully isolated and characterized an intermediate level of odor representation between olfactory nerve input and principal mitral/tufted cell output.

Optogenetic monitoring of membrane potentials.

Over the last decade, researchers in our laboratory have engineered and developed several series of genetically encoded voltage‐sensitive fluorescent proteins (VSFPs) by molecular fusion of a voltage‐sensing domain operand with different fluorescent reporter proteins. These genetically encoded VSFPs have been shown to provide a reliable optical report of membrane potential from targeted neurons and muscle cells in culture or in living animals. However, these various reporters also exhibit discrepancies in both their voltage‐sensing and targeting properties that are essentially related to the intrinsic characteristics of the fluorescent reporter proteins. It is therefore important carefully to select the sensor that is most appropriate for the particular question being investigated experimentally. Here we examine the current state of this subfield of optogenetics, address current limitations and challenges, and discuss what is likely to be feasible in the near future.