Phenotypic screening using waveform analysis of synchronized calcium oscillations in primary cortical cultures

At present, in vitro phenotypic screening methods are widely used for drug discovery. In the field of epilepsy research, measurements of neuronal activities have been utilized for predicting efficacy of anti-epileptic drugs (AEDs). Fluorescence measurements of calcium oscillations in neurons are commonly used for measurement of neuronal activities, and some anti-epileptic drugs have been evaluated using this assay technique. However, changes in waveforms were not quantified in previous reports. Here, we have developed a high-throughput screening system containing a new analysis method for quantifying waveforms, and our method has successfully enabled simultaneous measurement of calcium oscillations in a 96-well plate. Features of waveforms were extracted automatically and allowed the characterization of some anti-epileptic drugs using principal component analysis. Moreover, we have shown that trajectories in accordance with the concentrations of compounds in principal component analysis plots were unique to the mechanism of anti-epileptic drugs. We believe that an approach that focuses on the features of calcium oscillations will lead to better understanding of the characteristics of existing anti-epileptic drugs and allow prediction of the mechanism of action (MoA) of novel drug candidates.

Methods
Calcium imaging

A vial of Cal-520 (#21130, AAT-Bioquest) was resuspended to 2 µM in Tyrode’s buffer (0.1 mM Mg2+) containing 0.01% Pluronic F-127 (#P3000MP, Invitrogen). The cultures were washed three times in Tyrode’s buffer, and incubated in 80 µL of Cal-520 per well for 1 hour at 37°C. Culture plates were set to an assay stage and stabilized for more than 10 minutes before measurement.

For calcium imaging, an FDSS/µCell (Hamamatsu Photonics) kinetic plate reader was used. After incubation, 20 µL of compounds dissolved in Tyrode’s buffer were dispensed using the 96 dispenser head of the FDSS/µCell and, 5 or more minutes after dispensing, calcium signals were measured for 5 minutes using the following settings: exposure time 36.5 ms, excitation wavelength 480 nm, emission wavelength 540 nm, temperature controlled at 37°C.

Data analysis

Fluorescence intensity data were extracted using FDSS/µCell software. Data analysis and visualization were processed using the “Wave Finder” in Spotfire (Data Visualization & Analytics Software—TIBCO Spotfire, http://spotfire.tibco.com/) and custom code written in R for PCA. PCA features were selected based on correlation of features and visual inspection of each waveform.