title: A neuron-based screening platform for optimizing genetically-encoded calcium indicators.
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.
authors.affiliation: Genetically-Encoded Neuronal Indicator and Effector Project, Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, United States of America.
authors.forename: Trevor J
authors.initials: TJ
authors.lastname: Wardill
authors.forename: Tsai-Wen
authors.initials: TW
authors.lastname: Chen
authors.forename: Eric R
authors.initials: ER
authors.lastname: Schreiter
authors.forename: Jeremy P
authors.initials: JP
authors.lastname: Hasseman
authors.forename: Getahun
authors.initials: G
authors.lastname: Tsegaye
authors.forename: Benjamin F
authors.initials: BF
authors.lastname: Fosque
authors.forename: Reza
authors.initials: R
authors.lastname: Behnam
authors.forename: Brenda C
authors.initials: BC
authors.lastname: Shields
authors.forename: Melissa
authors.initials: M
authors.lastname: Ramirez
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Kimmel
authors.forename: Rex A
authors.initials: RA
authors.lastname: Kerr
authors.forename: Vivek
authors.initials: V
authors.lastname: Jayaraman
authors.forename: Loren L
authors.initials: LL
authors.lastname: Looger
authors.forename: Karel
authors.initials: K
authors.lastname: Svoboda
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Kim
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002478:Cells, Cultured; D004558:Electric Stimulation; D005453:Fluorescence; D017930:Genes, Reporter; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D018079:Receptors, GABA / Q000378:metabolism; D012996:Solutions
chemical_list: D007202:Indicators and Reagents; D018079:Receptors, GABA; D012996:Solutions; D018698:Glutamic Acid; D002118:Calcium