Calcium Imaging of AM Dyes Following Prolonged Incubation in Acute Neuronal Tissue
Fig 2
The impact of depolarization on calcium signals A) Bath application of KCl (10 mM) caused depolarization of the membrane potential of a layer 5 pyramidal neuron (somatosensory cortex), measured by whole-cell patch-clamp. B) Representative trace of a single cortical neuron shows spontaneous calcium transients followed by a large increase in dF/F following bath application of KCl (blue arrow). C) Plots of the average increase in calcium concentration in evoked cells from slices that were imaged <4 hrs (blue; n = 190 cells) and >24 hours (black, n = 236 cells) post slicing show similar kinetics. Measurements were aligned to the onset of KCl application. D) Bar graph depicting the percentage of evoked cells that recover after short local application of KCl (30 mM, 1 Sec; n = 4) or Glutamate (100 uM, 1 Sec; n = 4), in slices that were incubated for <4 hrs and >24 hrs. E) 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 slice recorded <4 hrs post slicing; Black trace–average calcium signal recorded in a slice >24 hrs post slicing. Red dots indicate the time points of local KCl application. F) Intracellular calcium signals following repetitive local application of Glutamate (100uM, 1 Sec). Grey–calcium traces in single cells; Blue–average calcium trace in a slice recorded <4 hrs post slicing; Black trace–average calcium signal recorded in a slice >24 hrs post slicing. Blue dots indicate the time points of Glutamate application.