Calcium Imaging of AM Dyes Following Prolonged Incubation in Acute Neuronal Tissue
Fig 4
Calcium dynamics in retinal slices A) Fluorescence image of the ganglion cell layer of a wholemount retina shows ubiquitous staining with Fura-2-AM (left image; 380nm excitation; 60x objective; scale bar 30 μm). Application of 30 mM KCl, caused a noticeable increase in 340/380nm ratio relative to F0 (dF/F). Middle–Spontaneously active cells; right–Evoked cells (30mM KCl). B) Quantification of percentage of cells that were spontaneously active or responded to 30mM KCl with an increase in dF/F (evoked) were not different between 4hrs and 24hrs(p>0.4; two tailed student t-test). C) Plots of the average increase in calcium concentration in evoked cells from slices that were imaged <4 hrs (blue; n = 118 cells) and >24 hours (black, n = 180 cells) post slicing show similar kinetics and are not statistically different (P>0.4; two tailed student t-test). Measurements were aligned to the onset of KCl application. D) Intracellular calcium signals following repetitive short term application of KCl (30 mM; 1 sec). Grey–calcium traces in single cells; Blue–average trace in a retina recorded <4 hrs post slicing; Black trace–average calcium signal recorded in >24 hrs post slicing. Red dots indicate the time points of KCl application. E) Box plot describing the median and range of the average fluorescent intensity of the spontaneous calcium signals. F) Bar graph depicting the average frequency (per min) of spontaneous calcium signals imaged following <4hrs and >24hrs post-slicing (p>0.7; two tailed student t-test).