Abstract
Since the discovery that astrocytes are characterized by Ca(2+)-based excitability, investigating the function of these glial cells within the brain requires Ca(2+) imaging approaches. The technical evolution from chemical fluorescent Ca(2+) probes with low cellular specificity to genetically encoded indicators (GECIs) has enabled detailed analysis of the spatial and temporal features of intracellular Ca(2+) 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) Ca(2+) imaging in astrocytes from the somatosensory cortex (SSCx) of adult mice in ex vivo acute cortical slices, performed using two genetically encoded Ca(2+) indicators, i.e., cytosolic GCaMP6f and endoplasmic reticulum-targeted G-CEPIA1er. 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-CEPIA1er. The analysis of Ca(2+) 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-CEPIA1er signal, which reveals metabotropic receptor activation as a dynamic decrease in free Ca(2+) 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 Ca(2+) levels and kinetics of Ca(2+) release. Key features • This protocol is useful to characterize basal and evoked Ca(2+) astrocyte activity in acute mouse brain slices, deepening analysis to different subcellular territories and compartments. • The induction of Ca(2+) 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 Ca(2+) experiments using epifluorescence or confocal microscopy.