Introduction

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Massive Ca²⁺ influx through NMDA receptor channels may be the primary mediator triggering glutamate-induced neuronal death. Neurons survive when challenged by NMDA in the absence of extracellular Ca²⁺ or in the presence of intracellular Ca²⁺ chelators (Choi, 1987; Tymianski et al., 1993); and the severity of glutamate neurotoxicity depends on the transmembrane Ca²⁺ gradient (Tymianski et al., 1993). A large rise in [Ca²⁺]_i resulting from Ca²⁺ influx, however, may not in itself be the primary determinant of subsequent cell death, and all Ca²⁺ influxes may not be equally neurotoxic (Tymianski et al., 1993). Studies using high affinity fluorescent Ca²⁺ indicators (e.g., Fura-2, Indo-1) to measure [Ca²⁺]_i do not show a correlation between the magnitude of glutamate-induced [Ca²⁺]_i increase and degree of neurotoxicity (Tymianski et al., 1993; Wang and Thayer, 1996). Wang and Thayer (1996) showed that the amplitude of the glutamate-induced [Ca²⁺]_i increase reached a maximum at 30 µM glutamate, a nontoxic concentration with 5 min exposure, whereas maximal neurotoxicity was elicited at glutamate concentrations > 300 µM. Large rises in [Ca²⁺]_i evoked by non-NMDA glutamate receptor activation or by voltage-gated Ca²⁺ channels that were of equal magnitude to those induced by NMDA receptor activation did not result in extensive neuronal death (Tymianski et al., 1993). Hartley et al. (1993) and Eimerl and Schramm (1994) showed that glutamate-induced neurotoxicity correlates with Ca²⁺ load measured by ⁴⁵Ca²⁺ uptake, but not with [Ca²⁺]_i measured by Fura-2. The total amount of ⁴⁵Ca²⁺ taken up into cultured cerebellar neurons during toxic NMDA receptor activation is about 17 mM, which is 10,000 times the maximal measured [Ca²⁺]_i. These data suggest that most of the Ca²⁺ taken up through the NMDA receptor is probably sequestered by subcellular organelles. The only organelle capable of accumulating such massive amounts of Ca²⁺ is the mitochondrion.

In addition to serving as the neuron's primary source of energy, mitochondria act as important buffers of cytosolic Ca²⁺ (Werth and Thayer, 1994) and help to prevent excessive, prolonged elevation of [Ca²⁺]_i. The huge electrochemical proton gradient across the inner mitochondrial membrane, generated by active extrusion of protons along the electron transport chain, provides a strong driving force for Ca²⁺ uptake through a Ca²⁺-selective uniporter. Mitochondrial Ca²⁺ uptake is sensitive to physiological changes in [Ca²⁺]_i, is rapid and reversible, and has a huge capacity (Sparagna et al., 1995; Jou et al., 1996; Trollinger et al., 1997; Babcock et al., 1997). Inhibitors that disrupt mitochondrial Ca²⁺ uptake severely compromise clearance of cytosolic Ca²⁺ after an imposed elevation (White and Reynolds 1995, 1997; Wang and Thayer 1996), leading to a huge increase in peak [Ca²⁺]_i and prolonged recovery time to base-line. Several studies indicate that mitochondrial Ca²⁺ uptake plays a prominent role in buffering the large Ca²⁺ load induced by intense glutamate receptor stimulation, and Ca²⁺ entry into mitochondria may account for the poor correlation between glutamate-induced neurotoxicity and glutamate-induced changes in [Ca²⁺]_i (Wang and Thayer 1996; White and Reynolds 1995, 1997; Khodorov et al., 1996).

Excessive Ca²⁺ influx into mitochondria may result in mitochondrial dysfunction (Schinder et al., 1996). Prominent, sustained mitochondrial depolarization follows intense NMDA receptor stimulation, and closely parallels the incidence of neuronal death (Schinder et al., 1996; White and Reynolds, 1996). Impaired ATP production (Wang et al., 1994), increased generation of reactive oxygen species (Coyle and Puttfarcken, 1993), and other detrimental processes (e.g., opening of the permeability transitional pore) have also been suggested to result from Ca²⁺-mediated mitochondrial injury after NMDA receptor stimulation. Substantial Ca²⁺ can be accumulated in mitochondria as a result of overloading the matrix with Ca²⁺; this disrupts the structural and functional integrity of the organelle (Garthwaite and Garthwaite, 1986). Hence, mitochondria may be critical intracellular targets of injury after intense NMDA receptor stimulation (White and Reynold, 1996) and, in this way, may act as a plausible link between massive Ca²⁺ influx and glutamate neurotoxicity (Schinder et al., 1996).

Still, the unique relationships between NMDA receptor activation, cytosolic Ca²⁺ fluxes, and mitochondrial Ca²⁺ uptake remain obscure. Do mitochondria take up Ca²⁺ at the same rate, regardless of which cytosolic Ca²⁺ influx pathway is activated? To explore this question, we loaded cultured striatal neurons with two fluorescent Ca²⁺ indicators, calcium green and rhod-2, to independently and simultaneously monitor cytosolic and mitochondrial Ca²⁺ changes in the same cell. After challenges with NMDA, kainate, KCl, or ionomycin, we examined and compared cytosolic and mitochondrial Ca²⁺ transients to determine the selectivity of mitochondrial Ca²⁺ uptake in response to different sources of cytosolic Ca²⁺ influx. Our results suggest that Ca²⁺ entering through NMDA receptors has "privileged" access to mitochondria.

	Materials and Methods

Top Summary Introduction Materials & Methods Results & Discussion References

Primary striatal neuronal culture. Striata from brains of Sprague-Dawley rat pups on embryonic day 17 were dissected and dissociated as described previously (Peng et al. 1998). Dissociated cells were plated onto 25-mm round glass coverslips (no. 1) precoated with poly-D-lysine (0.5 mg/ml, overnight) at a density of 110,000 cells per coverslip and maintained in 10% FBS/DMEM media in the incubator with 5% CO₂ at 37°. To enhance the survival rate of this low density neuronal culture, coverslips plated with confluent glial cells were inverted and placed above the coverslips plated with neurons. In this bilaminar system, the two coverslips were separated by wax pegs so as not to have any direct contact between the two coverslips. Two days after plating, cells were treated with 2 mM cytosine arabinoside (Ara-C) in 2.5% FBS/N2/DMEM medium (DMEM media with 2.5% FBS and 10% N2.1 nutrient) for the next 2 days to inhibit the growth of astrocytes. The culture media was then replaced by serum-free N2/DMEM medium. Neurons were maintained in this medium for up to 16 days in culture. The neurons employed for experiments were generally cultured for 9-12 days.

Fluorescent Ca²⁺ indicator loading. Glass coverslips with striatal neurons were rinsed with HEPES-buffered salt solution (containing 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂, 10 mM glucose, 10 mM HEPES, pH 7.4), then incubated in buffer solution containing 2 µM rhod-2/AM (Molecular Probes, Eugene, OR) at 25° for 6-10 min. Coverslips were rinsed again, then reincubated in buffer solution containing 5 µM calcium green 1N/AM (Molecular Probes) at 37° for 40-50 min. The coverslips were rinsed again and maintained at room temperature in the buffer solution for an additional 20 min before the experiment.

Laser scanning confocal microscopy. Glass coverslips with dye-loaded cells were mounted in a perfusion chamber and secured on the stage of a Zeiss Axioskop microscope equipped with the Insight Point laser scanning confocal system (Meridian Instruments). Neurons were sequentially excited by the 488-nm line of an argon laser (for calcium green) and the 568-nm line of a krypton laser (for rhod-2) through a dual band dichroic mirror. The emitted fluorescence signals from single neurons were filtered sequentially through a 530/30-nm band pass filter (for calcium green) and a 605-nm long pass filter (for rhod-2) mounted on a rotating wheel and were detected by an intensified charge-coupled device camera. The confocal pinhole aperture ranged from 80 to 135 µm. A 100× oil immersion objective (1.4 NA) was used in all experiments. The fluorescence signals were digitized and images were stored in random access memory. The time taken to acquire an image ranged from 0.15 to 0.4 sec. Each set of fluorescence images was recorded at a rate of 0.5 Hz. Each neuron studied represents a separate experiment using a separate culture well and coverslip. Control experiments using singly labeled cells demonstrated that our excitation and detection scheme allowed no cross-talk between fluorophore-detection pairings. That is, the calcium green signal was not detected in the rhod-2 channel, and the rhod-2 signal was not detected in the calcium green channel.

Kinetic data analysis. As described previously (Peng et al., 1998), the rhod-2 signal is highly localized in mitochondria, and as described below, the calcium green signal is seen diffusely throughout the cytoplasm. Therefore, to improve the specificity and signal-to-noise ratio of our measurements, calcium green signals from predefined cytosolic areas and rhod-2 signals from predefined mitochondrial areas in single neurons were used for measurement of signal intensity. Fluorescence intensities at a given time point were normalized to base-line values (expressed as F/F₀, where F₀ is the base-line value) and plotted against time. In some experiments, to better compare the time courses of the calcium green and rhod-2 fluorescence intensity changes, fluorescence was normalized and expressed as a percentage of the maximal increase [F(%max) = ((F  F_base)/(F_peak  F_base)) × 100%]. These normalized data were fitted to single exponential equations that yielded rate constants (K) from which t_1/2 values were calculated according to the equation t_1/2 = 0.693/K. The t_1/2 values were used to compare the rates of increase in fluorescence intensity within and across experiments.

Stimulating agents. NMDA, kainate, and ionomycin (a calcium ionophore), were added in HEPES-buffered salt solution. In the KCl experiment, the HEPES buffer contained 50 mM KCl and 95 mM NaCl, maintaining the original ionic strength and osmolarity.

Statistics. For each stimulus (e.g., NMDA), the t_1/2 values for cytosolic and mitochondrial Ca²⁺ transients were calculated simultaneously for all replicate experiments by fitting the data to single exponential equations using the Prism software package for Macintosh (GraphPAD Software, San Diego, CA). The fits to the exponential equations of cytosolic versus mitochondrial fluorescence changes for each stimulus were compared by two-tailed t tests. Comparison of fits across experiments (e.g., comparing the cytosolic t_1/2 for each stimulus) was by analysis of variance with post hoc Bonferroni tests.

	Results and Discussion

Top Summary Introduction Materials & Methods Results & Discussion References

Concurrent imaging of cytosolic and mitochondrial Ca²⁺ with calcium green and rhod-2. Most of the published studies on the role of mitochondria in buffering cytosolic Ca²⁺ loads have employed a single fluorescent Ca²⁺ indicator, such as Fura-2 or Indo-1, to monitor changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i). These Ca²⁺ indicators can easily get into the cell and distribute not only in the cytosol, but also into subcellular organelles, depending on loading temperature and loading time. Changes in the fluorescence signal from these Ca²⁺ indicators in response to Ca²⁺ increases reflect changes in both the cytosol and the organelles into which the indicators have partitioned. In this study, to reliably, independently, and simultaneously detect cytosolic and mitochondrial Ca²⁺ concentration changes in the same cell, we used two fluorescent Ca²⁺ indicators, calcium green and rhod-2. Unlike most fluorescent Ca²⁺ indicators that carry negative charges, rhod-2/AM (cell membrane-permeable form) contains one net positive charge. This unique chemical feature allows it to accumulate selectively into the highly negatively charged mitochondrial matrix. Once inside the matrix, mitochondrial esterases cleave the AM ester to liberate rhod-2 (membrane-impermeable free acid form). However, when loaded at warm temperature (37°), cytosolic esterases are active enough that the AM esters are cleaved before rhod-2/AM can enter mitochondria. This can lead to substantial cytosolic loading (unpublished observations). At cooler loading temperatures, when enzymatic activity is slowed, the fluorophore esters can reach the mitochondria before being hydrolyzed. We loaded rhod-2/AM into striatal neurons at room temperature to favor mitochondrial loading, and used a low concentration of the indicator (1-2 µM), and a short loading time (5-10 min) to minimize cytosolic loading. With this loading technique, we consistently obtain strong mitochondrial rhod-2 signal and weak cytosolic rhod-2 signal (Fig. 1A). We have previously published a more detailed account of the characteristics and selectivity of rhod-2 in monitoring mitochondrial Ca²⁺ concentration changes (Peng et al. 1998).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Confocal fluorescence images of a striatal neuron loaded with two Ca2+ indicators, rhod-2 and calcium green. (Refer to Materials and Methods for dye loading procedure and imaging technique.) A, Rhod-2 image. Rhod-2 fluorescence signals reside in highly localized, punctate areas which have been demonstrated to be mitochondria. B, Calcium green image of the same neuron. Calcium green fluorescence signals show a more uniform distribution. C, Superimposed rhod-2 and calcium green images. The intensity of the fluorescence signal is indicated by the pseudo-color bar to the right in each image. Scale bar, 5 µm. In a typical experiment, circular regions of interest, 1-2 µm in diameter, were drawn in a mitochondrial region as defined by high signal in the rhod-2 image (A) and a cytosolic region defined by uniform calcium green signal and lack of rhod-2 signal (e.g., green areas in C). Signal responses from these predefined regions of interest were used to generate the data in this report.

Source specificity for fast mitochondrial Ca²⁺ uptake. Previous studies on glutamate- (or NMDA-) induced Ca²⁺ load buffering by mitochondria have focused on changes in the magnitude of the peak [Ca²⁺]_i transient and the recovery time to base-line [Ca²⁺]_i levels that result from mitochondrial Ca²⁺ uptake inhibition (White and Reynold, 1995, 1997; Wang and Thayer, 1996; Khodorov et al., 1996). That there is a much greater Ca²⁺ load induced by glutamate compared with KCl-induced membrane depolarization was suggested by the observation that inhibition of mitochondrial Ca²⁺ uptake markedly potentiated the peak [Ca²⁺]_i and prolonged the recovery time after a glutamate challenge but not after depolarization. In these experiments, the effects of mitochondrial Ca²⁺ uptake inhibition were inferred from changes in [Ca²⁺]_i measured with a single fluorescence indicator, which primarily reflected cytosolic Ca²⁺ concentration ([Ca²⁺]_c) changes. With this method, [Ca²⁺]_m changes could not be observed directly.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Simultaneous monitoring of cytosolic and mitochondrial Ca2+ transients, as detected by changes in calcium green and rhod-2 fluorescence intensity, in single striatal neurons treated with (A) 100 µM NMDA, (B) 100 µM kainate, or (C) 50 mM KCl. Fluorescence intensity changes were normalized and expressed as F/F0 values, where F0 is the fluorescence intensity at base-line. Only one representative experiment from each treatment group is shown. The recovery time to return to base-line levels for NMDA-induced increases in cytosolic and mitochondrial Ca2+ are longer than those for kainate or KCl induced responses.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Initial rising phase of the cytosolic and mitochondrial Ca2+ responses. Normalized fluorescence data were fitted to exponential curves as described in the text. A, The cytosolic responses to NMDA, kainate and KCl were indistinguishable statistically. B, After NMDA application, the cytosolic and mitochondrial Ca2+ transients were virtually identical. C-E, After kainate, KCl, or ionomycin, the mitochondrial response lagged substantially behind the cytosolic response (p < 0.0001). Points, mean ± standard error of four independent experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   t1/2 values for cytoplasmic and mitochondrial Ca2+ transients induced by NMDA, kainate, KCl, and ionomycin. The cytosolic t1/2 values for the various stimuli did not differ statistically, nor did the cytosolic and mitochondrial responses to NMDA differ. In contrast, for kainate, KCl, and ionomycin, the mitochondrial t1/2 was substantially greater than the corresponding cytosolic response. Error bars, 95% confidence intervals; ***, p < 0.0001.

Potential mechanisms. Sparagna et al. (1995) demonstrated that Ca²⁺ can be sequestered into mitochondria by two distinct modes, a rapid uptake (high conductivity) mode of very short duration, followed by a slower uptake (low conductivity) mode, which has the characteristics of the uniporter. Could this rapid mode of mitochondrial Ca²⁺ uptake be responsible for the NMDA-induced fast mitochondrial Ca²⁺ uptake observed in this report? If so, why is this rapid uptake mode activated selectively by NMDA but not by kainate or membrane depolarization? Polyamines may provide the answer. Sparagna et al. (1995) showed that the rapid mode of mitochondrial Ca²⁺ uptake can be activated specifically by polyamines, especially spermine. Spermine and spermidine are selectively released from striatum by NMDA receptor activation, but not by kainate or quisqualate (Fage et al., 1992). Interestingly, Siddiqui and Iqbal (1994) observed that NMDA receptor-mediated ⁴⁵Ca²⁺ fluxes can be blocked by a polyamine synthesis inhibitor. Porcella et al. (1991) and Kish et al. (1991) showed that a polyamine synthesis inhibitor is neuroprotective against NMDA-induced brain damage in vivo. These observations suggest that the specificity of the NMDA-induced mitochondrial response may be dictated via second messenger activation. In this scenario, activation of NMDA receptors, but not other receptors, may increase local intracellular concentrations of polyamines that would then activate the rapid mode of mitochondrial Ca²⁺ uptake. This possibility is under investigation.