Analysis of neural activity with fluorescent protein biosensors

Abstract Genetically encoded voltage indicators (GEVIs) have emerged as a technology to optically record neural activity with genetic specificity and millisecond-scale temporal resolution using fluorescence microscopy. GEVIs have demonstrated ultra-fast kinetics and high spike detection fidelity in vivo, but existing red-fluorescent voltage indicators fall short of the response and brightness achieved by green fluorescent protein-based sensors. Furthermore, red-fluorescent GEVIs suffer from incomplete spectral separation from green sensors and blue-light-activated optogenetic actuators. We have developed Ace-mScarlet, a red fluorescent GEVI that fuses Ace2N, a voltage-sensitive inhibitory rhodopsin, with mScarlet, a bright red fluorescent protein (FP). Through fluorescence resonance energy transfer (FRET), our sensor detects changes in membrane voltage with high sensitivity and brightness and has kinetics comparable to the fastest green fluorescent sensors. Ace-mScarlet’s red-shifted absorption and emission spectra facilitate virtually complete spectral separation when used in combination with green-fluorescent sensors or with blue-light-sensitive sensors and rhodopsins. This spectral separation enables both simultaneous imaging in two separate wavelength channels and high-fidelity voltage recordings during simultaneous optogenetic perturbation.

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Erasable labeling of neuronal activity using a reversible calcium marker

eLife ◽

10.7554/elife.57249 ◽

2020 ◽

Vol 9 ◽

Author(s):

Fern Sha ◽

Ahmed S Abdelfattah ◽

Ronak Patel ◽

Eric R Schreiter

Keyword(s):

Neural Activity ◽

Calcium Concentration ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Light Exposure ◽

Time Windows ◽

Active Neuron ◽

Neuron Populations ◽

New Type ◽

The Brain

Understanding how the brain encodes and processes information requires the recording of neural activity that underlies different behaviors. Recent efforts in fluorescent protein engineering have succeeded in developing powerful tools for visualizing neural activity, in general by coupling neural activity to different properties of a fluorescent protein scaffold. Here, we take advantage of a previously unexploited class of reversibly switchable fluorescent proteins to engineer a new type of calcium sensor. We introduce rsCaMPARI, a genetically encoded calcium marker engineered from a reversibly switchable fluorescent protein that enables spatiotemporally precise marking, erasing, and remarking of active neuron populations under brief, user-defined time windows of light exposure. rsCaMPARI photoswitching kinetics are modulated by calcium concentration when illuminating with blue light, and the fluorescence can be reset with violet light. We demonstrate the utility of rsCaMPARI for marking and remarking active neuron populations in freely swimming zebrafish.

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Efficiency of cell-type specific and generic promoters in transducing oxytocin neurons and monitoring their neural activity during lactation

Scientific Reports ◽

10.1038/s41598-021-01818-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Keerthi Thirtamara Rajamani ◽

Amanda B. Leithead ◽

Michelle Kim ◽

Marie Barbier ◽

Michael Peruggia ◽

...

Keyword(s):

Gene Expression ◽

Neural Activity ◽

Viral Vectors ◽

Fluorescent Protein ◽

Viral Vector ◽

Female Rats ◽

Cell Populations ◽

Specific Cell ◽

Functional Studies ◽

Drive Gene

AbstractHypothalamic oxytocin (OXT) and arginine-vasopressin (AVP) neurons have been at the center of several physiological and behavioral studies. Advances in viral vector biology and the development of transgenic rodent models have allowed for targeted gene expression to study the functions of specific cell populations and brain circuits. In this study, we compared the efficiency of various adeno-associated viral vectors in these cell populations and demonstrated that none of the widely used promoters were, on their own, effective at driving expression of a down-stream fluorescent protein in OXT or AVP neurons. As anticipated, the OXT promoter could efficiently drive gene expression in OXT neurons and this efficiency is solely attributed to the promoter and not the viral serotype. We also report that a dual virus approach using an OXT promoter driven Cre recombinase significantly improved the efficiency of viral transduction in OXT neurons. Finally, we demonstrate the utility of the OXT promoter for conducting functional studies on OXT neurons by using an OXT specific viral system to record neural activity of OXT neurons in lactating female rats across time. We conclude that extreme caution is needed when employing non-neuron-specific viral approaches/promoters to study neural populations within the paraventricular nucleus of the hypothalamus.

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Fluorescent proteins for in vivo imaging, where's the biliverdin?

Biochemical Society Transactions ◽

10.1042/bst20200444 ◽

2020 ◽

Vol 48 (6) ◽

pp. 2657-2667

Author(s):

Felipe Montecinos-Franjola ◽

John Y. Lin ◽

Erik A. Rodriguez

Keyword(s):

Hydrogen Peroxide ◽

In Vivo Imaging ◽

Near Infrared ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Fluorescent Imaging ◽

Infrared Light ◽

Red Fluorescent Protein ◽

Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

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