Calcium Imaging of AM Dyes Following Prolonged Incubation in Acute Neuronal Tissue

Abstract

Calcium-imaging is a sensitive method for monitoring calcium dynamics during neuronal activity. As intracellular calcium concentration is correlated to physiological and pathophysiological activity of neurons, calcium imaging with fluorescent indicators is one of the most commonly used techniques in neuroscience today. Current methodologies for loading calcium dyes into the tissue require prolonged incubation time (45-150 min), in addition to dissection and recovery time after the slicing procedure. This prolonged incubation curtails experimental time, as tissue is typically maintained for 6-8 hours after slicing. Using a recently introduced recovery chamber that extends the viability of acute brain slices to more than 24 hours, we tested the effectiveness of calcium AM staining following long incubation periods post cell loading and its impact on the functional properties of calcium signals in acute brain slices and wholemount retinae. We show that calcium dyes remain within cells and are fully functional >24 hours after loading. Moreover, the calcium dynamics recorded >24 hrs were similar to the calcium signals recorded in fresh tissue that was incubated for <4 hrs. These results indicate that long exposure of calcium AM dyes to the intracellular cytoplasm did not alter the intracellular calcium concentration, the functional range of the dye or viability of the neurons. This data extends our previous work showing that a custom recovery chamber can extend the viability of neuronal tissue, and reliable data for both electrophysiology and imaging can be obtained >24hrs after dissection. These methods will not only extend experimental time for those using acute neuronal tissue, but also may reduce the number of animals required to complete experimental goals.

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).