Two-photon (2P) Microscopy to Study Ca<sup>2+</sup> Signaling in Astrocytes From Acute Brain Slices

Since the discovery that astrocytes are characterized by Ca²⁺-based excitability, investigating the function of these glial cells within the brain requires Ca²⁺ imaging approaches. The technical evolution from chemical fluorescent Ca²⁺ probes with low cellular specificity to genetically encoded indicators (GECIs) has enabled detailed analysis of the spatial and temporal features of intracellular Ca²⁺ signal. Different imaging methodologies allow the extraction of distinct information on calcium signals in astrocytes from brain slices, with resolution ranging from cell populations to single cells up to subcellular domains. • Here, we describe 2-photon laser scanning microscopy (2PLSM) Ca²⁺ imaging in astrocytes from the somatosensory cortex (SSCx) of adult mice in ex vivo acute cortical slices, performed using two genetically encoded Ca²⁺ indicators, i.e., cytosolic GCaMP6f and endoplasmic reticulum-targeted G-CEPIA1<i>er</i>. The main advantage of the 2PLSM technique, compared to single-photon microscopy, is the possibility to go deeper in the tissue while avoiding photodamage, by limiting laser excitation to a single focal plane. The fluorescent signal of the indicator is analyzed offline in different compartments-soma, proximal processes, and microdomains-for GCaMP6f experiments and in the perinuclear, somatic area for G-CEPIA1<i>er</i>. The analysis of Ca²⁺ signal from different compartments, although not providing a value of absolute concentration, allows a critical comparison of the degree of astrocyte activation between different experimental conditions or mouse models. Moreover, the analysis of G-CEPIA1<i>er</i> signal, which reveals metabotropic receptor activation as a dynamic decrease in free Ca²⁺ in the endoplasmic reticulum (ER), can provide information on possible alterations in this critical second messenger pathway in astrocytes, including, for example, steady-state ER Ca²⁺ levels and kinetics of Ca²⁺ release. Key features • This protocol is useful to characterize basal and evoked Ca²⁺ astrocyte activity in acute mouse brain slices, deepening analysis to different subcellular territories and compartments. • The induction of Ca²⁺ probe expression requires surgical experience in mice and appropriate stereotaxic equipment for adeno-associated viral (AAV) vector injection. • The imaging experimental protocol takes approximately 8 h from the beginning of brain slice preparation to completion of 2PLSM imaging. • The described protocol, from slice preparation to signal analysis, can also be adapted for astrocyte Ca²⁺ experiments using epifluorescence or confocal microscopy.