Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation

Grimley, Joshua S.; Li, Li; Wang, Weina; Lei, Wen; Beese, L.S.; Hellinga, Homme W.; Augustine, George J

doi:10.1523/jneurosci.4616-11.2013

Cited by 99 publications

(152 citation statements)

References 45 publications

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“…Wimmer et al (47) provided fluorometric data on [Cl − ] i changes in vivo by using SuperClomeleon (48), which has a somewhat higher affinity (20-40 mM) to Cl − than Clomeleon. This study has important differences from our approach, since they could only measure bulk changes of [Cl − ] i concentration in a relatively large brain volume.…”

Section: Discussion Properties and Advantages Of Lssmclophensor In Nementioning

confidence: 99%

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Sato

Artoni

Landi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

127

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Intracellular chloride ([Cl−] i ) and pH (pH i ) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl − ] i and pH i , but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl − and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl − ] i and pH i at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl − ] i and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic twophoton excitation protocol to simultaneously determine the changes of pH i and [Cl − ] i in response to hypercapnia and seizure activity.I ntracellular ion concentrations are controlled by plasmalemmal transporters and channels, which generate and dissipate ionic electrochemical gradients, respectively (1). In recent years, regulation of the intracellular Cl − concentration ([Cl − ] i ) in neurons has attracted lots of attention, because it is the main ion that carries current across GABA A (and also, glycine) receptors. Changes in [Cl − ] i exert an immediate effect on the reversal potential of GABAergic currents (E GABA ) and, thereby, on the properties of GABA A receptor-mediated transmission (2-4). The "ionic plasticity" of GABAergic signaling involves not only the passive flux of Cl − ions through membrane channels but also, a number of ion transporters that regulate [Cl − ] i . Furthermore, this mechanism is under the control of intracellular signaling cascades that regulate the expression patterns as well as functional properties of ion transporters and channels (5, 6). With regard to long-term ionic modulation of GABAergic transmission, a case in point is the decrease in [Cl − ] i that is generally thought to take place during maturation of most central neurons. According to this widely accepted scenario, the Na-K-2Cl cotransporter NKCC1 accumulates Cl − in immature neurons, thereby promoting depolarizing GABA responses (3, 7-9), which is followed by developmental upregulation of the neuron-specific K-Cl cotransporter KCC2 that is required for the generation of classical hyperpolarizing inhibitory postsynaptic potentials (IPSPs) (10).A wealth of electrophysiological evidence dating back to the work in vivo by Eccles and coworkers (11) has provided evidence for active regulation of [Cl − ] i in mammalian central neurons and its crucial effect on the driving force of Cl − in inhibitory synapses (1). However, thus far, there are no direct data on neuronal [Cl − ] i measured in vivo at the single-cell level in the living brain, and for in...

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Section: Discussion Properties and Advantages Of Lssmclophensor In Nementioning

confidence: 99%

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Sato

Artoni

Landi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

127

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“…Further improvement in EYFP/H148Q was made by random mutagenesis in the vicinity of the halide-binding site, with an additional V163S mutation lowering the half maximal inhibitory concentration (IC 50 ) for Cl -down to 40 mM (Galietta et al 2001). Although the H148Q mutation may improve chloride affinity, it also diminishes YFP fluorescence intensity (Elsliger et al 1999), thus it does not necessarily improve the signal-to-noise ratio (SNR) of chloride measurements (Grimley et al 2013).…”

Section: Yellow Fluorescent Protein and Its Variantsmentioning

confidence: 99%

“…When expressed by itself in vitro, Topaz has a K d for chloride of 163 mM at pH 7.1 (Grimley et al 2013). Topaz has the halide affinity sequence of F − > I − > Br − > Cl − .…”

Section: Yellow Fluorescent Protein and Its Variantsmentioning

confidence: 99%

Optogenetic Sensors for Monitoring Intracellular Chloride

2015

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“…however, it is challenging to use clomeleon to image gaBa-induced cl − fluxes, as the affinity of clomeleon (~ 100 mM) is far beyond the typical physiological range of intracellular cl − (~5 ~ 6 mM) and extracellular cl − in the synaptic cleft (as high as 4 mM) for postsynaptic inhibitory transmission [216]. To solve this issue, grimley et al tuned the halide affinity and fluorophore characteristics of yFP to develop a second generation of cl − indicator, Superclomeleon [217]. Superclomeleon has a cl − affinity of 8.1 mM and can be used in mouse neurons to images changes in cl − concentration associated with exogenously applied gaBa or inhibitory synaptic activity [217].…”

Section: Chloride Indicatorsmentioning

confidence: 99%

“…To solve this issue, grimley et al tuned the halide affinity and fluorophore characteristics of yFP to develop a second generation of cl − indicator, Superclomeleon [217]. Superclomeleon has a cl − affinity of 8.1 mM and can be used in mouse neurons to images changes in cl − concentration associated with exogenously applied gaBa or inhibitory synaptic activity [217].…”

Section: Chloride Indicatorsmentioning

confidence: 99%

Fluorescent Proteins for Neuronal Imaging

Zhao

Campbell

2015

Biological and Medical Physics, Biomedical Engineering

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over the past two decades, the growing selection of engineered fluorescent proteins have helped drive a revolution in the ability of researchers to image protein localization and biochemical dynamics in live cells in real time. although the fluorescent proteins were long preceded by other fluorophores compatible with live cell imaging, the fact that fluorescent proteins are fully genetically encoded has enabled them to be applied in applications that would not otherwise be possible. in particular, fluorescent proteins have enabled the creation of transgenic animals in which specific neuronal cell types are uniquely and fluorescently labeled. Furthermore, through the use of highly engineered fluorescent proteins that change their fluorescence in response to a change in calcium ion concentration or membrane potential, fluorescent proteins have enabled high resolution minimally invasive imaging of neuronal activity in model organisms. in this chapter we will provide an overview of fluorescent protein technology and detail the technological developments that have made such experiments possible. Particular emphasis will be placed on the development of strategies for engineering ca 2+ and voltage indicators, and the latest breakthroughs in these directions will be highlighted.

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Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation

Cited by 99 publications

References 45 publications

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Optogenetic Sensors for Monitoring Intracellular Chloride

Fluorescent Proteins for Neuronal Imaging

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