Genetically encoded probes for optical imaging of brain electrical activity.
ABSTRACT The combination of optical imaging methods with targeted expression of protein-based fluorescent probes constitutes a powerful approach for functional analysis of selected cell populations within intact neuronal circuitries. Herein, we lay out the conceptual motivation for optogenetic recording of brain electrical activity using genetically encoded voltage-sensitive fluorescent proteins (VSFPs), describe how the current generation of VSFPs has evolved, and demonstrate how VSFPs report membrane voltage signals in isolated cells, brain slices, and living animals. We conclude with a critical appraisal of VSFPs for voltage recording and highlight promising applications of this emerging methodology for bridging cellular and intact systems biology.
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ABSTRACT: Biological systems present multiple scales of complexity, ranging from molecules to entire populations. Light microscopy is one of the least invasive techniques used to access information from various biological scales in living cells. The combination of molecular biology and imaging provides a bottom-up tool for direct insight into how molecular processes work on a cellular scale. However, imaging can also be used as a top-down approach to study the behavior of a system without detailed prior knowledge about its underlying molecular mechanisms. In this review, we highlight the recent developments on microscopy-based systems analyses and discuss the complementary opportunities and different challenges with high-content screening and high-throughput imaging. Furthermore, we provide a comprehensive overview of the available platforms that can be used for image analysis, which enable community-driven efforts in the development of image-based systems biology.Cell Communication and Signaling 04/2013; 11(1):24. · 5.09 Impact Factor
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ABSTRACT: To better understand the connectivity of the brain, it is important to map both structural and functional connections between neurons and cortical regions. In recent years, a set of optogenetic tools have been developed that permit selective manipulation and investigation of neural systems. These tools have enabled the mapping of functional connections between stimulated cortical targets and other brain regions. Advantages of the approach include the ability to arbitrarily stimulate brain regions that express opsins, allowing for brain mapping independent of behavior or sensory processing. The ability of opsins to be rapidly and locally activated allows for investigation of connectivity with spatial resolution on the order of single neurons and temporal resolution on the order of milliseconds. Optogenetic methods for functional mapping have been applied in experiments ranging from in vitro investigation of microcircuits, to in vivo probing of inter-regional cortical connections, to examination of global connections within the whole brain. We review recently developed functional mapping methods that use optogenetic single-point stimulation in the rodent brain and employ cellular electrophysiology, evoked motor movements, voltage sensitive dyes (VSDs), calcium indicators, or functional magnetic resonance imaging (fMRI) to assess activity. In particular we highlight results using red-shifted organic VSDs that permit high temporal resolution imaging in a manner spectrally separated from Channelrhodopsin-2 (ChR2) activation. VSD maps stimulated by ChR2 were dependent on intracortical synaptic activity and were able to reflect circuits used for sensory processing. Although the methods reviewed are powerful, challenges remain with respect to finding approaches that permit selective high temporal resolution assessment of stimulated activity in animals that can be followed longitudinally.Frontiers in Neuroscience 01/2013; 7:54.
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ABSTRACT: Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC 50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC 50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo.Frontiers in Molecular Neuroscience 01/2013; 6:11.