A neuron-based screening platform for optimizing genetically-encoded calcium indicators.
PLoS One
; 8(10): e77728, 2013.
Article
em En
| MEDLINE
| ID: mdl-24155972
ABSTRACT
Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cálcio
/
Genes Reporter
/
Sinalização do Cálcio
/
Neurônios
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
/
Screening_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
PLoS One
Assunto da revista:
CIENCIA
/
MEDICINA
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Estados Unidos