Confocal Imaging and k-Means Clustering of GABAB and mGluR Mediated Modulation of Ca2+ Spiking in Hippocampal Neurons.

Abstract

Imaging cytosolic calcium in neurons is emerging as a new tool in neurological disease diagnosis, drug screening, and toxicity testing. Ca2+ oscillation signatures show a significant variation depending on GPCR targeting agonists. Quantification of Ca2+ spike trains in ligand induced Ca2+ oscillations remains challenging due to their inherent heterogeneity in primary culture. Moreover, there is no framework available for identification of optimal number of clusters and distance metric to cluster Ca2+ spike trains. Using quantitative confocal imaging and clustering analysis, we show the characterization of Ca2+ spiking in GPCR targeting drug-treated primary culture of hippocampal neurons. A systematic framework for selection of the clustering method instead of an intuition-based method was used to optimize the cluster number and distance metric. The results discern neurons with diverse Ca2+ response patterns, including higher amplitude fast spiking and lower spiking responses, and their relative percentage in a neuron population in absence and presence of GPCR-targeted drugs. The proposed framework was employed toshow that theclustering pattern of Ca2+ spikingcan be controlledusing GABAB and mGluR targeting drugs. This approach can be used for unbiased measurement of neural activity and identification of spiking population with varying amplitude and frequencies, providing a platform for high-content drug screening.