Two-photon (2P) Microscopy to Study Ca2+ Signaling in Astrocytes From Acute Brain Slices

Abstract

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-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 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-CEPIA1er 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.

Keywords: 2PLSM imaging; Acute brain slice; Astrocytes; Ca2+ signaling; G-CEPIA1er; GCaMP; Intracerebral AAV injection.

Figure 1.. Preparation for stereotaxic intracortical injections.

(A) Stereotaxic apparatus, stereoscope, and surgical tools (arrows) are prepared under the BL2 hood. (B) Surgical tools, marker, suture thread, and 1 mL syringe with PBS. (C) Glass capillary with marked notches is positioned on the stereotaxic holder. The inset shows the graph paper used for capillary labeling. (D) Anesthesia induction chamber. (E) Anesthesia vaporizer. (E) The mouse head is fixed in the stereotaxic apparatus, the brain is exposed, and the viral mix-loaded capillary is ready to be lowered for injection (yellow arrow points to the drilled site).

Figure 2.. 2-photon imaging of intracellular Ca²⁺ signal in astrocytes.

(A) Net for slices. (B) Brain slices in the beaker with the net, in standard solution under O₂/CO₂ bubbling. (C) Peristaltic pump for recording solution perfusion. (D) Perfusion chamber with a brain slice under the platinum grid in the 2P microscope setup, under a 20× objective. (E) Representative 2P images (average projection of 180 frames) of two SSCx astrocytes expressing GCamp6f and tdTomato. (F) Representative 2P images (average projection of 180 frames) of three SSCx astrocytes expressing CEPIA1er and tdTomato. Scale bar, 10 μm.