Ca2+ handling was analyzed using a combination of patch-clamp recordings, ratiometric Ca2+ imaging, and the ‘added buffer approach’. (A) Ca2+ resting level. The concentration of free cytosolic Ca2+ was increased in DIO mice. (B–G) Ca2+-handling properties. (B) fura-2 loading curve. POMC neurons were loaded via the patch pipette with the ratiometric Ca2+ indicator fura-2, which also serves as the added Ca2+ buffer. Fura-2 fluorescence was acquired at 360 nm excitation (isosbestic point of fura-2) every 30 s, and converted into fura-2 concentrations. (C) Decay kinetics of voltage-induced Ca2+ transients of the POMC neuron in (B). The images (left panels) were acquired at times indicated in (B) and demonstrate the increasing fura-2 concentration during loading. The graphs (right panels) demonstrate the effect of increasing added Ca2+ buffer (fura-2) concentrations on the decay kinetics of voltage-evoked Ca2+ transients. (C and D) Analysis of endogenous Ca2+-handling parameters in a single cell. With increasing fura-2 concentrations, the amplitudes of transients decreased, and the time constants (τtransient) for decay were prolonged (C and D). The decay time constants were plotted against the Ca2+-binding ratios of fura-2 (κB) (D). κB was calculated from the intracellular fura-2 concentration, the Kd of fura-2, and the resting concentration of free intracellular Ca2+. The solid line represents the linear fit to the data. An estimate of κS was obtained as the negative x-axis intercept. The Ca2+ extrusion rate is estimated from the slope of the fit and the endogenous decay time constant from the intercept with the y-axis. (E) The decay time constants of all recorded neurons were plotted as a function of κB for all POMC neurons of the control and DIO mice. The best linear fits with 95% confidence bands are shown. (F and G) To estimate the variance of the endogenous Ca2+-binding ratio (κS) and the extrusion rate (γ), we used a bootstrap method (1000 samples), which provided bootstrap distributions (n = 1000) of the parameters for the control and DIO mice. Vertical lines indicate the means. (F) Distributions of κS. Sixteen counts for the control and one count for the DIO cohort between 2000 and 7500 are not shown. (G) Distribution of γ. Eighteen counts for the control and one count for the DIO cohort between 400 and 1350 are not shown. Subsequently, the distributions were log-transformed to bring them closer to a Gaussian, before applying unpaired t-tests. ***p<0.001.