Elucidating regulators of astrocytic Ca²⁺ signaling via multi‐threshold event detection (MTED)

Abstract

Recent achievements in indicator optimization and imaging techniques promote the advancement of functional imaging to decipher complex signaling processes in living cells, such as Ca2+ activity patterns. Astrocytes are important regulators of the brain network and well known for their highly complex morphology and spontaneous Ca2+ activity. However, the astrocyte community is lacking standardized methods to analyze and interpret Ca2+ activity recordings, hindering global comparisons. Here, we present a biophysically‐based analytical concept for deciphering the complex spatio‐temporal changes of Ca2+ biosensor fluorescence for understanding the underlying signaling mechanisms. We developed a pixel‐based multi‐threshold event detection (MTED) analysis of multidimensional data, which accounts for signal strength as an additional signaling dimension and provides the experimenter with a comprehensive toolbox for a differentiated and in‐depth characterization of fluorescence signals. MTED was validated by analyzing astrocytic Ca2+ activity across Ca2+ indicators, imaging setups, and model systems from primary cell culture to awake, head‐fixed mice. We identified extended Ca2+ activity at 25°C compared to 37°C physiological body temperature and dissected how neuronal activity shapes long‐lasting astrocytic Ca2+ activity. Our MTED strategy, as a parameter‐free approach, is easily transferrable to other fluorescent indicators and biosensors and embraces the additional dimensionality of signaling activity strength. It will also advance the definition of standardized procedures and parameters to improve comparability of research data and reports.

Astrocytes and Microglia Exhibit Cell-Specific Ca2+ Signaling Dynamics in the Murine Spinal Cord

“…This opposite findings between ex vivo and in vivo recordings may be due to an activation of astrocytes and microglia resulting from the excessive manipulation required for spinal cord extraction and acute slice preparation resulting in an excessively high (astrocytes) or excessively low (microglia) Ca 2+ activity in line with the change of microglia morphology to a more amoeboid appearance in slices. It was recently shown for the brain that astroglial Ca 2+ dynamics differ between ex vivo and in vivo (as well as in situ ) recordings ( Müller et al, 2021 ) supporting our results. Astroglial ROA densities decrease in vivo along the imaging sessions, possibly hinting that higher ROA densities are associated with an alteration of astroglial signaling in the acute phase after laminectomy non-detectable by reactive markers.…”

Serotonin Receptor 5-HT2A Regulates TrkB Receptor Function in Heteroreceptor Complexes

“…To evaluate possible functional consequences of TrkB–5-HT 2A heterodimerization, we first assessed Ca 2+ activity in N1E-115 cells expressing TrkB-YPet, 5-HT 2A -mTurquoise2, or both. To this end, we used the Ca 2+ indicator GCaMP6f [ 48 ] and analyzed Ca 2+ dynamics with the multi-threshold event detection approach [ 49 ]. The ratio of the GCaMP6f fluorescent signal (F) to the saturated Ca 2+ signal (F max ) indicated that basal Ca 2+ levels were significantly higher following heterodimerization compared to control cells ( Figure 3 ).…”

Astrocyte heterogeneity and interactions with local neural circuits

“…Despite (these) major advances in our understanding of Ca 2+ signal types, and the mechanisms underlying their generation, their physiological significance and information content are not well understood -although recent work suggests that reducing the amplitude and duration of Ca 2+ signals in striatal astrocytes leads to excessive self-grooming in mice [82], while impairing Gi-coupled Ca 2+ signaling brain wide leads to impaired spatial memory [83]. The introduction of next generation signal processing tools [84][85][86], capable of accurately describing the dynamics of (subcellular) Ca 2+ transients, will undoubtedly help link the appearance of specific astrocytic Ca 2+ signals to specific functional outputs. This will have to be allied to improvements in imaging to capture the true spatial dynamics of the signal (3D imaging) [87], over extreme time domains extending from milliseconds to seconds [88].…”