- Research article
- Open Access
Calcium signals can freely cross the nuclear envelope in hippocampal neurons: somatic calcium increases generate nuclear calcium transients
© Eder and Bading; licensee BioMed Central Ltd. 2007
- Received: 29 January 2007
- Accepted: 30 July 2007
- Published: 30 July 2007
In hippocampal neurons, nuclear calcium signaling is important for learning- and neuronal survival-associated gene expression. However, it is unknown whether calcium signals generated by neuronal activity at the cell membrane and propagated to the soma can unrestrictedly cross the nuclear envelope to invade the nucleus. The nuclear envelope, which allows ion transit via the nuclear pore complex, may represent a barrier for calcium and has been suggested to insulate the nucleus from activity-induced cytoplasmic calcium transients in some cell types.
Using laser-assisted uncaging of caged calcium compounds in defined sub-cellular domains, we show here that the nuclear compartment border does not represent a barrier for calcium signals in hippocampal neurons. Although passive diffusion of molecules between the cytosol and the nucleoplasm may be modulated through changes in conformational state of the nuclear pore complex, we found no evidence for a gating mechanism for calcium movement across the nuclear border.
Thus, the nuclear envelope does not spatially restrict calcium transients to the somatic cytosol but allows calcium signals to freely enter the cell nucleus to trigger genomic events.
- Hippocampal Neuron
- Nuclear Envelope
- Calcium Signal
- Calcium Release
- Calcium Transient
The compartmentalization of eukaryotic cells into membrane-delineated organelles spatially restricts molecules and allows specialized functions that require different biochemical microenvironments to be carried out simultaneously. To regulate and coordinate metabolic activities, mechanisms have evolved that allow information transfer across compartment borders. Many signaling pathways activated in the cytosol upon stimulation from the environment impinge on targets in the cell nucleus and regulate gene expression. In neurons, transcriptional responses induced by electrical activity are critical for long-lasting adaptive responses such as information storage, memory formation, or the activation of pro-survival programs [1–4]. Calcium is the principal second messenger that couples neuronal activity to gene regulation . Several calcium-activated pathways can transmit signals to the nucleus. These include the ERK-MAP kinase and the p38 MAP kinase pathways, and a signaling pathway activated by the serine/threonine phosphatase, calcineurin (reviewed in ). However, the primary signal transducer is calcium itself that can propagate information from the site of signal generation at the plasma membrane into the nucleus [2, 7]. Electrical activity-induced increases in the nuclear calcium concentration are required for CREB- and CBP-mediated gene expression [8–10]. Moreover, nuclear calcium signaling is critical for the long-lasting synaptic plasticity and learning , and induces the expression of a genomic pro-survival program [12, 13]. Nuclear calcium transients in neurons are likely triggered by increases in the calcium concentration in the somatic cytosol. Although the nuclear envelope can restrict the exchange of molecules between the cytosol and the nucleoplasm, ions may enter and exit the nucleus via the nuclear pore complexes (NPCs). Precisely how nuclear calcium signals are generated and whether or not cytosolic calcium transients can freely cross the nuclear border is controversial [14, 15]. In the mouse pituitary cell line, AtT20, and in a variety of primary neurons, electrical activity-induced somatic calcium signals appear to spread readily to the nucleus [8, 10, 16–18]. In contrast, in HeLa cells, neuroblastoma cells, and primary rat sensory neurons, the nucleus may be insulated from cytosolic calcium transients [19, 20]. In addition, in Xenopus laevis oocytes, the filling state of intracellular calcium stores may regulate the conformational state of the NPC, which can affect diffusion of molecules between the cytosol and the nucleoplasm [21, 22]. It is particular important to understand the dynamics of calcium signaling across the nucleo-cytoplasmic border in hippocampal neurons, where neuronal activity-induced nuclear calcium transients control a neuroprotective gene expression program [12, 13] and are critical for learning and memory . Here, we used laser-assisted photolysis of caged calcium compounds to investigate the properties of the nucleo-cytoplasmic exchange of calcium in hippocampal neurons. Our analysis revealed that calcium diffuses freely into and out of the nucleus with no apparent impediment at the nuclear envelope. We found no evidence for a gating mechanism for calcium movement through the NPC. Thus, in hippocampal neurons, calcium waves towards the cell soma do not face a detectable barrier at the nuclear compartment border.
Release of calcium in spatially distinct regions using laser-assisted photolysis of caged calcium compounds
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).
Long-range calcium signaling within compartments and across the nuclear compartment border
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
How signals cross intracellular compartment borders is a fundamental question in biology. In this study we analyzed the mechanism of calcium signal propagation across the nuclear envelope in hippocampal neurons.
Nuclear calcium: key signal for adaptive responses in neurons
Neurons use changes in the intracellular calcium concentration to communicate signals generated by synaptic activity at the cell surface to the transcription-regulating machinery in the cell nucleus. Experiments in which either nuclear calcium was buffered  or the complex of calcium with calmodulin, the principal calcium sensor , was blocked specifically in the cell nucleus show that nuclear calcium signaling is a key mediator of neuronal gene expression [8–10], survival [12, 13], and learning . Given the central role of nuclear calcium in the control of adaptive responses, it is important to understand the precise mechanism of how calcium enters the cell nucleus and to uncover possible ways through which neurons can regulate this process to restrict access of calcium to the nucleus.
Calcium signals freely cross the nuclear envelope in hippocampal neurons
There is evidence to suggest that the nucleus is insulated from large increases in the cytosolic calcium concentration [19, 20]. The question arises as to what extend the nuclear membrane perforated by NPCs has an effect on particles of the size of around 0.2 nm, which corresponds to the atomic radius of calcium. NPCs are closely packed on the surface of the nuclear membrane . Given the diameter of the central transporter region together with the spoke-ring complex of the NPC of approximately 50 to 100 nm [28, 29] it is rather difficult to imagine how ion flux through the NPC could be restricted. A possible mechanism through which the cell could gate diffusion processes across the NPC has been suggested by Clapham and coworkers who identified by field emission scanning electron microscopy and atomic force microscopy a central plug in the NPC present in nuclear membrane preparations from Xenopus laevis oocyte . Under condition where intracellular calcium stores are depleted, the plug occluded the NPC channel and blocked diffusion of intermediate size molecules (about 10 kDa), suggesting that calcium stores may regulate the conformational state of the nuclear pore complex, and thereby passive diffusion of molecules between the cytosol and the nucleoplasm. However, smaller molecules (such as Lucifer yellow) or ions were shown to diffuse freely across nuclear membrane preparation and intact nuclei even after calcium store depletion. Consistent with the observations in Xenopus laevis oocyte, we find that even after intracellular calcium store depletion, calcium can freely cross the nuclear border. These results do not rule out the existence of a central nuclear pore complex plug in hippocampal neurons; however, they suggest that a gating mechanism for calcium flux across the nuclear envelope does not exist.
Possible other mechanisms for generating nuclear calcium signals
It remains an open question whether nuclear calcium transients can be generated without increases in the cytosolic calcium concentration. It is conceivable that calcium is directly released into the nucleoplasm from the inter-membrane space of the nuclear envelope, which is continuous with the endoplasmic reticulum (ER) [30, 31]. Such a mechanism would depend on calcium release channels, such as the IP3 receptor or the ryanodine receptor being localized to the inner nuclear envelope. There are no electron microscopy studies that unambiguously demonstrate the presence of IP3 receptors and/or ryanodine receptors in the inner nuclear envelope. However, biochemical and electrophysiological studies using non-neuronal cells and Purkinje neurons suggested the both IP3 receptors and ryanodine receptors may be present in the inner nuclear envelope [32–36]. Moreover, CD38/ADP-ribosyl cyclase, the enzyme that catalyses the conversion of nicotinamide adenine dinucleotide to cyclic adenosine diphosphate ribose (cADP-ribose), an agonist of the ryanodine receptor, is localized to the inner nuclear envelope . Ryanodine receptors, if present in the inner nuclear membrane and activated by cADP-ribose, could trigger calcium release into the nucleoplasm. However, to our knowledge signal-induced calcium transients that occur exclusively in the nucleus have not been observed in neurons. In addition, in HeLa cells, the analysis of spontaneous calcium events in over 700 cells failed to identify calcium signals that unambiguously originated in the cell nucleus , suggesting that HeLa cell nuclei are devoid of IP3 receptor- and/or ryanodine receptor-operated calcium stores. Our finding that photolysis-induced nuclear calcium transients are significantly smaller in hippocampal neurons after CPA-mediated calcium store depletion (see Figure 9F) are consistent with the existence of intra-nuclear sites of calcium release that may enhance nuclear calcium signaling. However, conditions under which hippocampal neurons generate nuclear calcium transients independently of cytosolic calcium increases remain to be identified.
Calcium, the primary signal transducer in neuronal activity-dependent transcription, diffuses into and out of the nucleus with no apparent impediment at the nuclear envelope. The nuclear compartment border does not spatially restrict activity-induced calcium transients to the somatic cytosol but allows calcium signals to freely enter the cell nucleus to trigger genomic events.
Cell culture and stimulations
Hippocampal neurons from new-born Sprague-Dawley rats were prepared as described  except that growth media was supplemented with B-27 (Invitrogen, Karlsruhe, Germany). Cells were plated on poly-D-lysine and laminin-coated glass coverslips. Calcium imaging and uncaging were done after a culturing period of 10–14 days. Bursts of action potential firing were induced by treatment of cultured hippocampal neurons with 50 μM bicuculline (MP Biomedicals, Heidelberg, Germany). The membrane of hippocampal neurons was depolarized by increasing the extra-cellular K+ concentration by 50 mM. Calcium release from intracellular stores was induced using bath application of 100 μM carbachol (Carbamylcholine chloride, Sigma-Aldrich Chemie GmbH, München, Germany). Depletion of intracellular calcium stores was achieved by treatment for 30 min with 30 μM cyclopiazonic acid (CPA, Tocris Bioscience, Bristol, UK), an inhibitor of the SR/ER Calcium-ATPase.
Loading procedure, calcium imaging, and [Ca2+] measurements
Cells were loaded with 5–15 μM NP-EGTA, AM (Invitrogen, Karlsruhe, Germany) by incubation at 37°C, 5% CO2 for 90 min. Afterwards, they were loaded with 3.6 μM Fluo-4, AM (Invitrogen, Karlsruhe, Germany) by incubation at room temperature in darkness for 25 min. Before starting imaging/uncaging cells were loaded with 200 nM MitoTracker Deep Red 633 (Invitrogen, Karlsruhe, Germany). Fluorescence images were obtained using a Leica SP2 confocal microscope with an HCX PL APO CS 40.0 × 1.25 NA OIL UV objective (Leica, Wetzlar, Germany). Cells mounted in a perfusion chamber (LIS, Reinach, Switzerland) were imaged at room temperature in a buffered salt-glucose-glycine (SGG) solution containing (mM) NaCl 140.1, KCl 5.3, MgCl2 1.0, CaCl2 2.0, Hepes 10.0, glycine 1.0, glucose 30.0, and sodium pyruvate 0.5 on the stage of an inverted Leica DMIRBE microscope (Leica, Wetzlar, Germany). Experiments at higher frame rate (11.6 Hz) were also done at 37°C. Fluo-4 was excited by a 488 nm laser line and emission was collected at 500–550 nm. MitoTracker Deep Red 633 was excited by a 633 nm laser line and emitted light was collected at 640–720 nm. Release of calcium from NP-EGTA was achieved by exposure of predefined regions to UV laser light at 364 nm and 351 nm over a period of several frames during the image acquisition. The total exposure time t is calculated by t = t pixel × n pixel × n frame (t pixel = time to scan one pixel, n pixel = number of pixels of the uncaging area, n frame = number of frames with UV exposure). In the uncaging experiments, images were taken every 401 msec or 86 msec; in the experiments with bicuculline and carbachol, images were taken every 1.6 sec. To calibrate the fluorescence signal (F), Fluo-4 was saturated by adding 50 μM ionomycin (F max , Sigma-Aldrich Chemie GmbH, München, Germany) to the perfusion solution and then quenched with MnCl2 (F min ). [Ca2+] was expressed as a function of the Fluo-4 fluorescence K d × [(F-F min )/(F max -F)] . The validity of calcium measurements critically depends on the accuracy of the calibration, particularly when calcium concentrations are being analyzed and compared in different cellular compartments . To avoid measurement artifacts, calibration was done pixel by pixel to relate the fluorescent values during calcium release with the correspondent values during the calibration. The time constants at half maximum, the tFmax/2 values, were determined by approximating the data to an exponential function of the form f(x) = exp(a+bx+cx2). The coefficients a, b and c were calculated with the standard procedure of a mathematical fit based on the Gaussian method of least squares. All fits were performed considering the values during the rise of the calibrated traces from baseline to peak calcium signal.
We thank Iris Bünzli-Ehret for preparing hippocampal cultures and C. Peter Bengtson for discussion and reading the manuscript. This work was supported by the Alexander von Humboldt Foundation (Wolfgang Paul Prize to HB), the German Research Foundation (DFG), and the Sonderforschungsbereich (SFB) 488.
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