PMID- 24155972
OWN - NLM
STAT- MEDLINE
DCOM- 20140617
LR  - 20211021
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 8
IP  - 10
DP  - 2013
TI  - A neuron-based screening platform for optimizing genetically-encoded calcium 
      indicators.
PG  - e77728
LID - 10.1371/journal.pone.0077728 [doi]
LID - e77728
AB  - 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.
FAU - Wardill, Trevor J
AU  - Wardill TJ
AD  - Genetically-Encoded Neuronal Indicator and Effector Project, Janelia Farm 
      Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, United 
      States of America.
FAU - Chen, Tsai-Wen
AU  - Chen TW
FAU - Schreiter, Eric R
AU  - Schreiter ER
FAU - Hasseman, Jeremy P
AU  - Hasseman JP
FAU - Tsegaye, Getahun
AU  - Tsegaye G
FAU - Fosque, Benjamin F
AU  - Fosque BF
FAU - Behnam, Reza
AU  - Behnam R
FAU - Shields, Brenda C
AU  - Shields BC
FAU - Ramirez, Melissa
AU  - Ramirez M
FAU - Kimmel, Bruce E
AU  - Kimmel BE
FAU - Kerr, Rex A
AU  - Kerr RA
FAU - Jayaraman, Vivek
AU  - Jayaraman V
FAU - Looger, Loren L
AU  - Looger LL
FAU - Svoboda, Karel
AU  - Svoboda K
FAU - Kim, Douglas S
AU  - Kim DS
LA  - eng
GR  - Howard Hughes Medical Institute/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20131014
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (Indicators and Reagents)
RN  - 0 (Receptors, GABA)
RN  - 0 (Solutions)
RN  - 3KX376GY7L (Glutamic Acid)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Action Potentials/physiology
MH  - Animals
MH  - Calcium/*metabolism
MH  - *Calcium Signaling
MH  - Cells, Cultured
MH  - Electric Stimulation
MH  - Fluorescence
MH  - *Genes, Reporter
MH  - Glutamic Acid/metabolism
MH  - Humans
MH  - Indicators and Reagents
MH  - Neurons/*metabolism
MH  - Rats
MH  - Receptors, GABA/metabolism
MH  - Solutions
PMC - PMC3796516
COIS- Competing Interests: The authors have the following competing interests. A patent 
      application has been filed on the neuronal culture screening methods 
      ("Genetically Encoded Calcium Indicators and Methods of Use" USPTO 61/711,995). 
      There are no further patents, products in development or marketed products to 
      declare. This does not alter the authors' adherence to all the PLOS ONE policies 
      on sharing data and materials, as detailed online in the guide for authors.
EDAT- 2013/10/25 06:00
MHDA- 2014/06/18 06:00
PMCR- 2013/10/14
CRDT- 2013/10/25 06:00
PHST- 2013/03/01 00:00 [received]
PHST- 2013/09/04 00:00 [accepted]
PHST- 2013/10/25 06:00 [entrez]
PHST- 2013/10/25 06:00 [pubmed]
PHST- 2014/06/18 06:00 [medline]
PHST- 2013/10/14 00:00 [pmc-release]
AID - PONE-D-13-09065 [pii]
AID - 10.1371/journal.pone.0077728 [doi]
PST - epublish
SO  - PLoS One. 2013 Oct 14;8(10):e77728. doi: 10.1371/journal.pone.0077728. 
      eCollection 2013.