2-Photon excitation calcium imaging

Cytosolic calcium levels from PVN^OT neurons conditionally tagged by Ai14-tdTomato within acute coronal brain slices (250 μm) of mice were monitored by 2-photon excitation microscopy using the genetically encoded calcium indicator GCaMP6f. Single brain slices were transferred to a chamber mounted on a stage of an upright multiphoton laser scanner microscope (FVMPE-RS system, Olympus) and continuously perfused with bubbled-aCSF using a gravity-driven perfusion system at a rate of ∼3 mL/min in the presence of synaptic blockers (20 μM CNQX, 50 μM D-AP5, and 100 μM picrotoxin). Neurons were visualized with a 25× water immersion objective. Excitation illumination was generated by an InSight X3 DUAL tunable laser system (Spectra-Physics). The FluoView image acquisition software (FV31S-SW, Olympus) was used to tune laser emission wavelength to 930 and 1045 nm in order to obtain 2-photon absorption signals from GCaMP6f and tdTomato fluorophores, respectively, at an acquisition rate of 0.5 Hz. Calcium imaging from PVN^OT neurons consisted of a 3-min baseline recording followed by bath application of CCK (50 nM) and drug washout. At the end of each experiment, 20 mM KCl was bath-applied to check neuronal viability and calcium signal integrity. Only neurons that responded to KCl were used for analysis. Calcium transients were estimated as changes in GCaMP6f-based fluorescence intensity over the baseline (ΔF/F₀), considering a calcium event when ΔF/F₀ > 3 standard deviations greater than the baseline fluorescence signal. The number of calcium events were then plotted over time grouped into 1-min bins in order to quantify changes in the frequency of calcium events in PVN^OT neurons in response to CCK application.