Fig 1.
Calcium imaging in acute brain slices A) Flow diagram illustrating the slicing and calcium AM dye loading protocol in brain slices. B) Fluorescence images of a neocortical slice shows ubiquitous staining of cortical neurons and glia with Fura-2-AM (left image; 380nm excitation; 20x objective; scale bar 100 μm). Application of 30 mM KCl, caused a noticeable increase in 340/380nm ratio relative to F0 (dF/F), depicting three classes of cells according to their staining pattern. Left–Loaded cells; Middle–Spontaneously active cells (imaged as ratiometric changes before the application of KCl) and Right–Evoked cells (following 30mM KCl). Red and blue correspond to high and low Ca2+ concentrations, respectively C) Bar graph depicting the percentage of spontaneous and evoked cells, out of the total loaded cells within the field of view indicating a slight yet insignificant decrease in the both spontaneous and evoked cells following >24 hours in the Braincubator (p>0.9; two tailed student t-test).
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.
Fig 3.
Spontaneous Calcium signals in brain slices A) Time lapse fluorescence microscopy images of slices loaded with Fluo-4 depicting spontaneous calcium transients. Top–Images from slice <4hrs post slicing; Bottom—Images from slice >24hrs post slicing. Arrows point to individual cells that show calcium transients (color coded). B) Sample traces of intracellular spontaneous calcium signals in cortical slices at the indicated times post slicing. C,D) Box plots of the average fluorescent intensity (C) and frequency (D) of the spontaneous calcium transients were not different at <4hrs and >24hrs post-slicing.
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).