Spectraphysics maitai

To monitor the cytoplasmic free Ca²⁺ concentraton ([Ca²⁺]_i) in situ, astrocytes of neocortical slices were loaded by 30 min incubation with 1 µM of Rhod-2AM or Oregon Green BAPTA-2 at 33°C. Two-photon imaging of neurons and astrocytes was performed using a Zeiss LSM-7MP multi-photon microscope coupled to a SpectraPhysics MaiTai pulsing laser; experiments were controlled by ZEN LSM software (Carl Zeiss, Germany). Images were further analysed off-line using ZEN LSM (Carl Zeiss) and ImageJ (NIH) software. The [Ca²⁺]_i levels were expressed as ΔF/F ratio averaged over region of interest (ROI). For analysis of spontaneous Ca²⁺-transients in astrocytes, three ROIs located at branches and one ROI located at the soma were chosen. Overall Ca²⁺ response to agonists of eCB agonist or synaptic stimulation was quantified using ROIs covering the whole cell image.

To monitor the cytoplasmic free Ca²⁺concentraton ([Ca²⁺]_i) in situ, astrocytes of neocortical slices were loaded via 30 min incubation with 1 μM of Rhod-2AM or Oregon Green Bapta-2AM and sulphorhodamine 101 (wild-type (WT) mice) at 33°C. Two-photon images of neurons and astrocytes were acquired at 5 Hz frame-rate using a Zeiss LSM-7MP multi-photon microscope coupled to a SpectraPhysics MaiTai pulsing laser; experiments were controlled by ZEN LSM software (Carl Zeiss, Germany). Images were further analyzed offline using ZEN LSM (Carl Zeiss) and ImageJ (NIH) software. The [Ca²⁺]_i levels were expressed as ΔF/F ratio averaged over a region of interest (ROI). For analysis of spontaneous Ca²⁺–transients in astrocytes, three ROIs located over dendrites and one ROI located over the soma were chosen. Overall Ca²⁺-response to receptors agonists or synaptic stimulation was quantified using an ROI covering the whole cell image.