Release of calcium in spatially distinct regions using laser-assisted photolysis of caged calcium compounds
Hippocampal neurons were loaded with both the calcium indicator Fluo-4 and the calcium cage nitro-phenyl-EGTA (NP-EGTA) to release calcium in spatially distinct regions while monitoring calcium signals propagating throughout the cell (Figure 1A, B, and 1C). The local activation of NP-EGTA was achieved by exposure to ultraviolet (UV) laser light from a confocal laser scanning microscope. The scanning system allowed UV exposure with a rapid time course and high spatial precision, and thus produced calcium release in a defined area in a temporally controlled manner.
We first established the conditions to control calcium release. We exposed several cells in a field of view to UV light varying the exposure time (Figure 2A) as well as the UV laser power (Figure 2B). In both cases the calcium signal rose immediately after switching on the UV light. The amplitude of the calcium signal increased with the duration of the UV exposure; the rise time of the calcium transients decreased with the UV laser power. Furthermore, the amount of released calcium could be controlled by changing the area exposed to the UV light (data not shown). UV exposure to cells loaded only with Fluo-4 caused no change in the calcium concentration (Figure 2C). The UV exposure time was calculated taking into account that the activation of NP-EGTA occurred pixel by pixel over a period of several frames during the image acquisition (Figure 1D and 1E). Thus, the total exposure time t is calculated by t = t
pixel
× n
pixel
× n
frame
where t
pixel
is the time which is needed to scan one pixel, n
pixel
is the number of pixels of the uncaging area, and n
frame
the number of frames with UV exposure. The duration over which uncaging occurred d can be calculated by d = n
frame
/freq
frame
where freq
frame
is the imaging frequency.
We next selected a tool to precisely position the uncaging spot in the region of interest. Because we were interested in properties of calcium transients in different cellular compartments and calcium crossing the nuclear envelope (NE), we needed a dye that would allow us to distinguish nucleoplasm and cytoplasm. This dye should penetrate easily the plasma membrane without harming the cells and should not interfere with the uncaging procedure or calcium imaging. We chose the mitochondrion-selective stain MitoTracker Deep Red 633. Loading neurons with MitoTracker and the DNA specific stain HOECHST 33258 showed that MitoTracker was excluded from the nucleus (Figure 3A). For subsequent experiments, hippocampal neurons were loaded with MitoTracker, NP-EGTA, and Fluo-4. Calcium imaging experiments, in which hippocampal networks were either stimulated with the GABAA receptor blocker bicuculline (which triggers periodic and synchronous action potential bursting; [10, 23]) or exposed to elevated extra-cellular K+ concentrations (causing membrane depolarization), demonstrated that MitoTracker does not interfere with calcium signaling (Figure 3B and 3C). Closer examination of calcium signals induced by action potential firing revealed, at an image acquisition rate of 0.6 Hz, virtually synchronous time courses of the nuclear and cytoplasmic calcium transients (Figure 3D).
The cell loading procedure consisted of an incubation period with NP-EGTA for 90 min at 37°C and 5% CO2, followed by a 30-minute incubation period with the calcium indicator Fluo-4 acetoxymethyl (AM) ester at room temperature to avoid compartmentalization of the dye [24]. During subsequent deesterification of the AM ester the hippocampal neurons were treated with MitoTracker for a further 15 min. The loaded cells were mounted in a perfusion chamber and the uncaging spot was defined (Figure 4B and 5B). Calcium was released by UV light and Fluo-4 fluorescent changes were measured (Figure 4C and 5C). The total UV exposure time of an area of 5 μm2 and 25 frames was ~10 msec. This was sufficient to evoke maximal calcium release from the caged compound. We considered four different conditions for our analysis of calcium propagation within cellular compartments and across the NE: uncaging in the nucleus and calcium diffusion both within the nucleus and across the NE (Figure 4); uncaging in the cytoplasm (near the nuclear border) and calcium diffusion both within the cytoplasm and across the NE (Figure 5). Calcium signals were measured at regions equidistant from the centre of the uncaging area (Figure 4D and 4E, Figure 5D and 5E). A summary of the quantitative analysis of all calcium imaging traces measured at the various distances from the uncaging spot within a compartment and across the nuclear envelope is shown in Figure 8.
Generation and propagation of nuclear calcium signals
We first uncaged caged calcium in the nucleus. The signals were measured at a 2 μm and 5 μm distance from the uncaging spot inside and outside the nucleus. Irrespective of the presence of the nuclear border between the uncaging spot and the area of calcium measurements, the calcium signals rose with virtually identical slopes to the same maximum values (Figure 4D and 4E; see also summary of calcium imaging data in Figure 8C). To confirm that calcium diffused radially, we measured the signal at various points at 2 μm and 5 μm distances from the uncaging spot, determined the maximum values, and calculated by an exponential fit the time, tFmax/2, required to reach half of the maximum value. The tFmax/2 values obtained were: 13.4 ± 0.2 sec (n = 5) and 13.5 ± 0.2 sec (n = 5) in the nucleus at distances of 2 μm and 5 μm, respectively, from the uncaging spot, and 12.6 ± 0.1 sec (n = 5) and 13.7 ± 0.2 sec (n = 8) in the cytoplasm at distances of 2 μm and 5 μm, respectively, from the uncaging spot. The signal increases of the calibrated traces from basal to maximum were 0.92 ± 0.04 and 1.10 ± 0.13 at the 2 μm distance and 0.94 ± 0.08 and 0.89 ± 0.21 at the 5 μm distance for the nuclear and cytoplasmic signals, respectively. The differences in the values obtained for the 2 μm distance may be due to the close proximity of the region of calcium measurement and the site of calcium release. Because of the small size of the UV spot, a higher power of UV laser light (~2.4 mW) was required to obtain a sharp rise in calcium. It is conceivable that under those conditions, scattered light of the UV laser beam affected the calcium indicator in the immediate vicinity of the uncaging area. Visualizing the data in one-dimensional profiles at different time points confirmed that calcium spread equally throughout the cell. The calcium signals were similar on both sides of the NE at the resting ('basal') or the elevated state ('UV peak'). Even during the period of decay ('decay'), differences in the amplitudes of nucleoplasmic and cytoplasmic calcium signals could not be detected (Figure 4F). The virtually identical magnitudes and kinetics of signals at 2 μm and 5 μm as well as intermediate and larger diffusion distances (data not shown) indicate that calcium propagation out of the nucleus is not measurably reduced or slowed by the nuclear membrane.
Generation and propagation to the nucleus of cytosolic calcium signals
It is possible that the process of calcium leaving the nucleus differs from that of calcium entering the nucleus. To test this, we shifted the uncaging spot to the cytosol and measured calcium propagation into the nucleus. To uncage amounts of calcium comparable to our nuclear uncaging experiments, we chose the same conditions (i.e. identical spot size, UV laser power and exposure time). The results were very similar to those obtained after nucleoplasmic calcium release. Signals were measured at a 6 μm and 10 μm distance from the centre of the uncaging spot in the cytoplasm and the nucleus. In addition, we monitored calcium signals at the border between nucleus and cytoplasm (Figure 5D and 5E; see also summary of calcium imaging data in Figure 8D). The nucleoplasmic signal increased as fast as the cytoplasmic signal and as fast as the signal in the perinuclear space at both the 6 μm and 10 μm distances (tFmax/2 = 11.6 ± 0.3 sec (n = 17), 11.6 ± 0.3 sec (n = 13) and 11.5 ± 0.4 sec (n = 7) at the 6 μm distance for the cytoplasm, nuclear border and nucleus, respectively; tFmax/2 = 11.7 ± 0.3 sec (n = 4), 11.8 ± 0.3 sec (n = 5) and 11.7 ± 0.2 sec (n = 9) at the 10 μm distance for the cytoplasm, nuclear border and nucleus, respectively). The peak calcium signal in the cytoplasm was slightly higher than that in the nucleus, whereas the maximum value in the perinuclear space was, at least at the 6 μm distance, higher than the cytoplasmic and nuclear values (0.95 ± 0.36, 1.74 ± 0.91 and 0.78 ± 0.17 at the 6 μm distance for the cytoplasm, nuclear border and nucleus, respectively; 0.95 ± 0.17, 0.75 ± 0.13 and 0.84 ± 0.12 at the 10 μm distance for the cytoplasm, nuclear border and nucleus, respectively).
It remained possible that calcium measurements at a higher temporal resolution could reveal a partial barrier to calcium movement at the NE. We therefore repeated our experiments at higher frame rate (11.6 Hz) for both calcium signals propagating into and out of the nucleus. We achieved faster image acquisition by narrowing the field of view of the scanning system to 512 × 32 pixels (Figure 6A and 6C). Measurements of calcium signals at distances from the uncaging spot of 3 μm and 7 μm in the nucleus and the cytoplasm did not reveal any delay of calcium signals crossing the NE (Figure 6B and 6D; see also summary of all calcium imaging data in Figure 8E and 8F). For nuclear calcium release the tFmax/2 values were 3.7 ± 0.2 sec (n = 3) in the nucleus and 3.7 ± 0.1 sec (n = 3) in the cytoplasm at a distance of 3 μm from the uncaging spot, and 4.0 ± 0.1 sec (n = 3) in the nucleus and 3.9 ± 0.1 sec (n = 3) in the cytoplasm at a distance of 7 μm from the uncaging spot. For cytoplasmic calcium release we obtained tFmax/2 = 3.1 ± 0.1 sec (n = 3) in the nucleus and 3.3 ± 0.1 sec (n = 3) in the cytoplasm at a distance of 3 μm from the uncaging spot, and tFmax/2 = 3.4 ± 0.1 sec (n = 3) in the nucleus and 3.4 ± 0.1 sec (n = 3) in the cytosol at a distance of 7 μm from the uncaging spot. A summary of all experiments performed at 11.6 Hz indicates that the kinetics of calcium signal propagation is independent of the direction of the calcium flow (i.e. into the nucleus vs. out of the nucleus) and not measurably affected by the presence of the NE (Figure 8E and 8F). Acquiring images at 11.6 Hz, we obtained similar results in experiments in which calcium signal propagation across the nuclear border was measured at 37°C (data not shown). Thus, calcium appears to diffuse freely between the cytosol and the nucleus and equilibrates quickly between the two compartments. Within the limits of the temporal resolution of our imaging experiments, the NE does not attenuate or slow down calcium movements and thus, does not represent a diffusion barrier for calcium in hippocampal neurons.
Long-range calcium signaling within compartments and across the nuclear compartment border
We further investigated possible differences between calcium signal spreading within one compartment and across the NE by placing the uncaging spot further away from the nucleus near a dendrite and measuring the cytoplasmic signal at several distances (Figure 7A and 7B). The time courses measured at 6 μm and 10 μm distances were virtually identical. We also acquired signals at a 13 μm distance from the cytosolic uncaging spot both in the nucleus and the cytoplasm (Figure 7C). The results obtained revealed that even at relatively distant regions the spreading of the calcium signal within a compartment and across the NE was similar. This property of calcium signal spreading appears to be independent of the calcium concentration and was also observed for smaller size photolysis-induced calcium transients (Figure 7D and 7E).
The nuclear envelope is not a barrier for calcium signal propagation
A quantitative analysis of the time constants at half maximum, tFmax/2 (calculated from exponential fits; see Figure 8A and 8B) from all imaging traces measured at the various distances from the uncaging spot confirmed that the presence of the NE in the path of calcium signal propagation does not lead to a measurable delay in signal spreading (Figure 8C, D, E and 8F). For calcium release in the nucleus acquiring images at 2.5 Hz, the time constants at half maximum, tFmax/2, at the 5 μm distance from the uncaging spot measured in the nucleus and cytosol were 13.7 ± 0.4 sec (n = 3) and 14.3 ± 0.4 sec (n = 3), respectively; at the 7 μm distance from the uncaging spot they were 13.7 ± 0.3 sec (n = 3) and 14.7 ± 0.7 sec (n = 3) in the nucleus and cytosol, respectively. The time constants for calcium release in the cytosol measured at the 5 μm distance from the uncaging spot in the nucleus and cytosol were 13.6 ± 1.0 sec (n = 3) and 13.5 ± 1.0 sec (n = 3), respectively; at the 8 μm distance from the uncaging spot they were 13.9 ± 1.0 sec (n = 3) and 13.8 ± 1.3 sec (n = 3) in the nucleus and cytosol, respectively (Figure 8C and 8D). Acquiring images at 11.6 Hz, the time constants for calcium release in the nucleus measured at the 3 μm distance from the uncaging spot were 3.2 ± 0.4 sec (n = 3) and 3.4 ± 0.1 sec (n = 3) in the nucleus and cytosol, respectively; measured at the 7 μm distance from the uncaging spot, they were 3.5 ± 0.2 sec (n = 3) and 3.6 ± 0.1 sec (n = 3) in the nucleus and cytosol, respectively. For calcium release in the cytoplasm they were 3.4 ± 0.2 sec (n = 3) in the nucleus and 3.4 ± 0.2 sec (n = 3) in the cytoplasm at the 3 μm distance from the uncaging spot, and 3.7 ± 0.1 sec (n = 3) in the nucleus and 3.6 ± 0.1 sec (n = 3) in the cytoplasm at the 7 μm distance from the uncaging spot (Figure 8E and 8F). For both image acquisition rates (i.e. 2.5 Hz and 11.6 Hz), we obtained, for the various distances from the uncaging spot, virtually identical mean values of tFmax/2 for calcium signal spreading within the compartment and calcium signal spreading across the NE (Figure 8C, D, E and 8F). The slightly wider distribution of the data obtained for calcium signals released in the cytoplasm may be due to a higher degree of variation of the uncaging-induced calcium events, which could be caused by calcium-induced-calcium-release (CICR) from intracellular calcium stores or by the uptake of calcium into intracellular stores.
Lack of evidence for gating mechanism for calcium signal propagation across the nuclear border
It has been suggested that diffusion of molecules across the NE is inhibited by the depletion of intracellular calcium stores [21, 22, 25]. We therefore investigated the possibility that calcium store depletion affects calcium movements into and out of the nucleus. ER calcium stores were emptied using the SR/ER calcium-ATPase inhibitor cyclopiazonic acid (CPA). Store depletion was confirmed by demonstrating that treatment of hippocampal neurons with the cholinergic agonist carbachol failed to generate calcium transients in CPA treated neurons (Figure 9A and 9B). Having depleted intracellular stores, we repeated our experiments of local uncaging to measure calcium diffusion from the nucleus towards the cytoplasm and vice versa. We found that upon UV light-induced calcium release in the nucleus or the cytoplasm, increases in calcium were detected on either site of the NE with virtually identical kinetics and amplitudes (Figure 9C, D, and 9E). For calcium release in the nucleus, the time constants tFmax/2 measured at the 5 μm distance from the uncaging spot were 14.8 ± 0.2 sec (n = 9) and 15.1 ± 0.4 sec (n = 15) in the nucleus and in the cytoplasm, respectively (Figure 9C). For cytosolic calcium release, tFmax/2 measured at the 7 μm distance from the uncaging spot were 16.5 ± 0.3 sec (n = 10) and 16.1 ± 0.4 sec (n = 9) in the nucleus and cytosol, respectively (Figure 9D). The rises of the calibrated traces from baseline to peak calcium signal in the nucleus and cytosol were 0.94 ± 0.09 and 0.77 ± 0.15, respectively, for calcium release in the nucleus and 0.85 ± 0.09 and 0.74 ± 0.16, respectively, for calcium release in the cytoplasm. Compared to control, the calcium signals obtained under conditions of calcium store depletion were smaller. For UV light-induced calcium release in the nucleus, the nuclear calcium and cytosolic calcium signals in CPA treated cells were 45 ± 3% (n = 4) and 61 ± 4% (n = 5), respectively, of those obtained in control conditions; for UV light-induced calcium release in the cytosol, the nuclear calcium and cytosolic calcium signals in CPA treated cells were 49 ± 7% (n = 4) and 66 ± 2% (n = 5), respectively, of those obtained in the control cells (Figure 9F). These results indicate that calcium release from intracellular stores contributes to the calcium transients observed following UV light-induced calcium release. However, calcium stores do not appear to be linked to a gating mechanism that could modulate calcium signal propagation across the nuclear border.