Imaging CO2-Evoked Calcium Signaling in BAG Neurons

Animals expressing a flp-17p::YC3.60 (yellow cameleon 3.60) transgene were used for ratiometric imaging of relative calcium concentration in BAG cell bodies (Bretscher et al., 2011 (link)). After immobilization, animals were placed under a microfluidic PDMS chamber and exposed to a 0% CO₂ (3 min) - X% CO₂ (3 min) - 0% CO₂ (3 min) stimulus train, with X corresponding to 1%, 3% or 5% CO₂ depending on the experiment. To measure CO₂-evoked tonic Ca²⁺ activity in BAG, the time interval for CO₂ stimulation was prolonged from 3 min to 18 min. In all experiments, the background O₂ level was 7% O₂. Calcium imaging was done at 2 frames/s on an AZ100 microscope (Nikon) bearing a TwinCam adaptor (Cairn Research) mounted with two ORCAFlash4.0 V2 digital cameras (Hamamatsu) using an AZ Plan Fluor 2x lens with 2x zoom and an exposure time of 500 ms.