Biosensing Motor Neuron Membran Potential i Live Zebrafish Embryoer
Summary
Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.
Abstract
The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos.
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).
Introduction
In vivo systemisk komponentanalyse giver forskere mulighed for at undersøge cellulær adfærd på den mest pålidelige måde. Dette er især tilfældet, når aktiviteten under kontrol er stærkt påvirket af cellecelleinteraktioner (både kontakt- og ikke-kontaktafhængige) som i nervesystemet, hvor membranspændingsændringer driver kommunikationen blandt spændende celler. Forståelsen af informationen kodet af disse elektriske signaler er nøglen til at forstå den måde nervesystemet virker i både fysiologiske og sygdomstilstande.
For at studere celle elektriske egenskaber i de mest ikke-invasive fysiologiske forhold er der for nylig udviklet flere genetisk kodede spændingsindikatorer 1 . I modsætning til de tidligere generationer af optiske spændingsfølere (primært spændingsfølsomme farvestoffer) 2 giver GEVI'er mulighed for in vivo analyser af det intakte neurale system ogDeres udtryk kan begrænses til specifikke celletyper eller populationer.
Zebrafish embryoet er det valgte in vivo "substrat" for at drage fordel af det store potentiale, der tilskrives GEVI'er. Takket være sin optiske klarhed og dets forenklede, men evolutionært konserverede nervesystem gør Zebrafish-modellen faktisk det muligt at identificere og manipulere hver enkelt cellulær komponent i et netværk. Faktisk har beskæftigelsen af den FRET-baserede GEVI Mermaid 3 ført til identifikation af præ-symptomatiske ændringer i spinalmotor-neuronadfærd i en zebrafiskmodel af amyotrofisk lateralsklerose (ALS) 4 .
Følgende in vivo- protokol beskriver hvordan man overvåger de elektriske egenskaber hos spinalmotorneuroner i intakte zebrafiskembryoner, der udtrykker havfrue på en neuronspecifik måde. Desuden demonstrerer det, hvordan farmakologisk induceret chanGes i sådanne elektriske egenskaber kan være forbundet med ændringer i hyppigheden af embryonale spontane coilings, den stereotype motoriske aktivitet, som karakteriserer zebrafiskens bevægelsesadfærd i meget tidlige udviklingsstadier.
Protocol
Representative Results
Discussion
Protokollen, der præsenteres her, tillod os at undersøge sammenhængen mellem de elektriske egenskaber af zebrafisk embryon spinalmotor neuroner og spontan coiling opførsel, den tidligste stereotypiske motor aktivitet, der vises omkring 17 hpf embryonale udvikling og varer indtil 24 hpf 10 .
Vores tilgang giver forskere et værktøj til at studere neurale systemet af intakte embryoner, der fuldt ud bevarer kompleksiteten af interaktionerne mellem celler i et …
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Simona Rodighiero for her priceless support with the FRET imaging analysis.
Materials
| Low Melting Point Agarose | Sigma-Aldrich | A9414 |
| DMSO | Sigma-Aldrich | W387520 |
| Riluzole | Sigma-Aldrich | R116 |
| Pfu Ultra HQ DNA polymerase | Agilent Technologies – Stratagene Products Division | 600389 |
| T3 Universal primer | Sigma-Aldrich | |
| Wizard SV Gel and PCR Clean-Up system | Promega | A9280 |
| Universal SmaI primer | Eurofins | |
| StrataClone Mammalian Expression Vector System / pCMV-SC blunt vector | Agilent Technologies – Stratagene Products Division | 240228 |
| SmaI | New England Biolabs | R0141S |
| T4 DNA ligase | Promega | M1801 |
| SalI | New England Biolabs | R0138S |
| EcoRV | New England Biolabs | R0195S |
| 35 mm, glass-bottomed imaging dish | Ibidi | 81151 |
| forceps | Sigma-Aldrich | F6521 |
| Stereomicroscope | Leica Microsystems | M10 F |
| Digital camera | Leica Microsystems | DFC 310 FX |
| Leica Application Suite 4.7.1 software | Leica Microsystems | |
| QuickTime Player, v10.4 | Apple | |
| Confocal microscope (inverted) | Leica Microsystems | TCS SP5 |
| Microinjector | Eppendorf | Femtojet |
| ImageJ macro Biosensor_FRET | ||
| GraphPad Prism 6.0c | GraphPad Software, Inc |
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