SIGAA – SIGnaling Automated Analysis: a new tool for Ca2+ signaling quantification using ratiometric Ca2+ dyes

Abstract

Astrocytes are non-neural cells, restricted to the brain and spinal cord, whose functions and morphology depend on their location. Astrocyte-astrocyte and astrocyte-neuron interactions occur through cytoplasmic Ca2+ levels changes, that are assessed to determine cell function and response (i.e. drug testing). Evaluation of changes in intracellular Ca2+ levels is mostly centered on fluorescence imaging approaches, performed through video recording of cells incubated with Ca2+-sensitive dyes. By observing ion concentration shifts over time in a delimited region of interest (ROI) comprising a single cell, it is possible to attain conclusions on cell responses to specific stimuli. Our work describes a tool named SIGAA – SIGnaling Automated Analysis, for astrocyte ROI-based fluorescent imaging, which is tailored for two wavelengths dyes by using two inputs of Ca2+ signaling recorded frames/videos and outputs a set of features relevant to the experiment’s conclusions and cell characterization. SIGAA performs automatic drift correction for the two recorded videos with a template matching algorithm, followed by astrocyte identification (ROI) using morphological reconstruction techniques. SIGAA then extracts intracellular Ca2+ evolution functions for all identified ROIs, detects function transients, and estimates a set of features for each signal, which are very similar to the ones obtained by the traditional methods and software used so far. SIGAA is a new fully automated tool, which can speed up hour-long studies and analysis to a few minutes, showing reliable results as the validity tests indicate.

Adenosine receptors are the on‐and‐off switch of astrocytic cannabinoid type 1 (CB1) receptor effect upon synaptic plasticity in the medial prefrontal cortex

“…The A 2A R activation lead to a significant decrease of CB1R elicited Ca 2+ transient amplitude (Ca 2+ T (ADA+CGS) : 1.24 ± 0.18 vs. Ca 2+ T (ADA) :1.47 ± 0.07, one‐way ANOVA: F (2,38) = 23.404, p = .0375) (Figure 2d1,d2,e), an effect that was counteracted by the selective antagonism of A 2A R, Scheme 58261 (50 nM) (Ca 2+ T (ADA+SCH+CGS) :1.42 ± 0.11), showing that A 2A R downregulates CB1R‐mediated Ca 2+ signaling in astrocytes. Furthermore, to investigate adenosinergic influence in Ca 2+ signaling kinetics, a computational analysis was employed previously described in literature (Lopes et al, 2023). Prior to CB1R activation, A 2A R activation was shown to elevate baseline Ca 2+ levels (Figure S3A) consistent with previous reports (Kanno & Nishizaki, 2012) while no significant differences were observed either in rise or decay slopes (Figure S3B,C) for all pharmacology used.…”