High-Throughput Analysis of Subcellular Ratiometric Imaging Data: Cyclic AMP in Astrocytes

Astrocytes, a highly heterogeneous neuroglial cell type, execute a myriad of homeostatic roles in the central nervous system. Although these cells are not firing action potential, they are excitable as demonstrated by an increase in the concentration of cytosolic second messengers, including free calcium concentration ([Ca2+]i) and cyclic adenosine monophosphate ([cAMP]i). In comparison with changes in [Ca2+]i that appear spatially and temporally heterogeneous in astrocytes, the first measurements in [cAMP]i in 2014 revealed that subcellular distribution is homogeneous and that changes in [cAMP]i determine the morphology of astrocytes following a bell-shaped curve dependency. A comparison of the subcellular distribution in [cAMP]i in arborized versus nonarborized astrocytes revealed changes in resting levels in [cAMP]i as well as differences in subcellular regions in resting and stimulated conditions. These measurements utilized image segmentation, which was time-consuming and cumbersome. This chapter presents a comprehensive study of MATLAB-based software designed to facilitate high-throughput analysis of ratiometric imaging data focusing on the dynamics of second messenger signaling, in particular calcium (Ca2+) and cyclic adenosine monophosphate (cAMP), within astrocytes. The SAMO (subcellular analysis of microdomain objects) software encompasses a user-friendly graphical interface, options for outlier removal and normalization, region of interest and spline selection, data visualization, and extensive data export capabilities. The usefulness of the tool is exemplified through a case study demonstrating effective visualization of cAMP levels in a single cell. With this solution, subcellular analysis of signaling activity captured by ratiometric images is greatly facilitated, empowering investigations of diverse signaling pathways and dynamic processes at the single-cell level, ultimately advancing our understanding of neurobiological phenomena.