Mapping of IP3-Mediated Ca2+ Signals in Single Human Neuroblastoma SH-SY5Y Cells: Cell Volume Shaping the Ca2+ Signal

K. van Acker, B. Bautmans, G. Bultynck, K. Maes, A. F. Weidema, P. de Smet, J. B. Parys, H. de Smedt, L. Missiaen, and G. Callewaert

Laboratory of Physiology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium


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van Acker, K., B. Bautmans, G. Bultynck, K. Maes, A. F. Weidema, P. de Smet, J. B. Parys, H. de Smedt, L. Missiaen, and G. Callewaert. Mapping of IP3-Mediated Ca2+ Signals in Single Human Neuroblastoma SH-SY5Y Cells: Cell Volume Shaping the Ca2+ Signal. J. Neurophysiol. 83: 1052-1057, 2000. Fast confocal laser-scanning microscopy was used to study spatiotemporal properties of IP3-mediated Ca2+ release signals in human SH-SY5Y neuroblastoma cells. [Ca2+]i increases were not affected by ryanodine (30 µM) or caffeine (10 mM) and largely insensitive to removal of external Ca2+, indicating predominance of IP3-induced Ca2+ release. Ca2+ signals evoked by high concentration (10 µM) of the muscarinic agonist carbachol appeared as self-propagating waves initiating in cell processes. At low carbachol concentrations (500 nM) Ca2+ changes in most cells displayed striking spatiotemporal heterogeneity. The Ca2+ response in the cell body was delayed and had a smaller amplitude and a slower rise time than that in processes. Ca2+ changes in processes either occurred in a homogeneous manner throughout the whole process or were sometimes confined to hot spots. Regional differences in surface-to-volume ratio appear to be critical clues that determine the spatiotemporal pattern of intracellular Ca2+ release signals.


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An increase in cytosolic Ca2+ concentration is a ubiquitous signal that can trigger or regulate a diversity of cellular activities including muscular contraction, neurotransmitter release, synaptic plasticity, cell proliferation, and cell apoptosis. In many cell types, agonists that stimulate the production of the second-messenger inositol 1,4,5-trisphosphate (IP3) increase the global intracellular Ca2+ concentration by releasing Ca2+ from IP3-sensitive stores (reviewed in Berridge 1997). It has been proposed that these global IP3-mediated Ca2+ signals arise from the synchronized activation of localized clusters of IP3Rs (Bootman and Berridge 1995) similar to those generated by ryanodine receptor activation in muscle cells (Cheng et al. 1993). Local IP3-mediated Ca2+ release signals have now been visualized and measured in a number of cells including Xenopus oocytes (Parker and Yao 1991; Sun et al. 1998; Yao et al. 1995), HeLa cells (Bootman et al. 1997), RBL cells (Horne and Meyer 1997), astrocytes (Yagodin et al. 1994), PC12 cells (Koizumi et al. 1999; Reber and Schindelholz 1996), vascular endothelial cells (Huser and Blatter 1997), cerebellar Purkinje neurons (Finch and Augustine 1998; Takechi et al. 1998), and cultured hippocampal neurons (Koizumi et al. 1999). The cellular mechanisms accounting for the spatial restriction of elementary Ca2+ release signals remain uncertain. Possible mechanisms include inhomogeneous spatial distribution of IP3Rs and the expression of different IP3R isoforms. Weak stimulation thus would only activate the most dense or sensitive sites and produce an elementary Ca2+ release event. Stronger stimulation would excite all release sites, causing a global Ca2+ signal (Bootman and Berridge 1995).

In the present study we have used fast laser-scanning microscopy to look for IP3-mediated Ca2+ release events in human SH-SY5Y neuroblastoma cells, which express only type I IP3R (Wojcikiewicz and Luo 1998). Stimulation of these cells with the muscarinic agonist carbachol (CCh) causes a transient rise in intracellular Ca2+ through IP3-mediated Ca2+ release (Lambert and Nahorski 1990; Murphy et al. 1991). Our findings demonstrate that these cells can generate spatially and temporally distinct Ca2+ signals. We further examined how geometric features contribute to the spatiotemporal properties of these Ca2+ signals.


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Cell culture and solutions

Experiments were performed on single cultured SH-SY5Y neuroblastoma cells (European Collection of Cell Cultures, ECACC 94030304). Cells were cultured in DMEM-HamF12 medium (GIBCO Laboratories), supplemented with 4 mM L-glutamine, 100 IU/ml penicillin-streptomycin, 1% nonessential amino acids, and 15% fetal calf serum at 37°C in a humidified atmosphere of 95% O2-5% CO2. Cells were plated on gelatin-coated coverslips at a density of 104 cells/coverslip and used between 5 and 13 days after plating. For experimentation cells were continuously perfused with a standard salt solution containing (in mM) 150 NaCl, 6 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.4. All experiments were performed at room temperature (20-22°C).

For rapid exchange of the extracellular solution or application of agonist, a multibarreled pipette with a common outlet placed at a distance of ~40 µm (Cell MicroControls, Virginia Beach, VA) from the cells under study was used. The dead space-time of the system was ~4 s.

In some experiments a nominally Ca2+-free saline supplemented with 2 mM EGTA was used. Ryanodine was purchased from Molecular Probes.

Confocal Ca2+ measurements

Neuroblastoma cells were loaded with the fluorescent Ca2+ indicator fluo-4 by incubating the cells in standard salt solution containing 10 µM of the acetoxymethyl (AM) ester of the dye (fluo-4 AM; Molecular Probes) for 30-45 min at room temperature. After loading, the cells were washed for at least 15 min with standard saline and transferred to a perfusion chamber on an upright microscope (Optiphot-2, Nikon) equipped with a ×40 water-immersion objective (N.A. 0.8, Zeiss). The microscope was attached to a confocal laser-scanning system (Odyssey, Noran Instruments). Fluo-4 was excited with the 488-nm line of an argon ion laser, and emitted fluorescence was measured at wavelengths >505 nm. Fluorescence images were acquired at 25 Hz (50-Hz frame rate after off-line deinterlacing) and digitized by a frame-grabber board connected to a personal computer (Kinetic Imaging, Liverpool, UK). Fluorescence changes were analyzed off-line by choosing small regions of interest (ROI). Background-corrected fluorescence traces were calculated as %Delta F/F, i.e., fluorescence increase divided by average prestimulus fluorescence. Contour images were reconstructed from the average of acquired images.

Under the present culturing conditions, cells bodies and processes of neighboring cells often made numerous contacts. To ascertain that Ca2+ signals in selected ROI originated from the same cell, kinetics of Ca2+ signals in all neighboring cells were carefully analyzed. The latency time of the CCh response varied greatly among cells making this dissection feasible. Only those cells, where the morphological overlay was well confined and the differences in Ca2+ response with adjacent cells were obvious, were further analyzed.


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Carbachol and caffeine responses in SH-SY5Y cells

When SH-SY5Y cells were exposed to 10 µM CCh for 5 s, nearly all cells (92%) exhibited a large and transient change in intracellular [Ca2+] (Fig. 1). To check whether the carbachol response involves Ca2+ release from IP3 or ryanodine-sensitive stores, SH-SY5Y cells were exposed to 10 mM caffeine. Figure 1 shows that in a small number of cells caffeine elicited a fast and large Ca2+ transient, but most cells failed to respond to caffeine. When those cells that failed to respond to caffeine were subsequently exposed to 10 µM CCh, nearly all cells responded, suggesting that their intracellular Ca2+ stores were not depleted before the caffeine challenge. Caffeine-sensitive cells generally also responded to CCh. For comparison, the CCh- and caffeine-induced Ca2+ signal in Fig. 1A were obtained in the same cell. Pretreatment of cells with 30 µM ryanodine had no effect on CCh-induced Ca2+ signals (data not shown). Removal of extracellular Ca2+ (data not shown) had no apparent effect on the amplitude of the CCh-induced Ca2+ transient but accelerated its relaxation. Subsequent reapplication of Ca2+ caused a secondary [Ca2+]i rise (data not shown) indicating that the decay of the Ca2+ transient is controlled by capacitive Ca2+ influx (Grudt et al. 1996) in concert with Ca2+ removal and reuptake mechanisms. The results thus indicate that the CCh response is predominantly mediated by Ca2+ release from IP3-sensitive stores. The slower onset and decay of the CCh response versus the caffeine response (Fig. 1A) can be accounted for by the intermediate steps required in the production and degradation of IP3.



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Fig. 1. Ca2+ signals evoked by 10 µM carbachol (CCh) or 10 mM caffeine in SH-SY5Y cells. A: the Ca2+ response of a single SH-SY5Y cell stimulated 1st with 10 mM caffeine (green trace) and then with 10 µM CCh (red trace). Between the 2 applications there was a 5-min wash out period. Solid bar indicates the time of drug application. B: 10 µM CCh caused a Ca2+ response in nearly all SH-SY5Y cells. By contrast, only a small population of CCh-sensitive cells responded to 10 mM caffeine. Data collected from 120 cells. C: comparison of the time course of CCh-induced Ca2+ changes in the cell body (green trace) and process (red trace). Contour image shows the corresponding regions over which the relative changes in fluorescence were integrated. The kinetics and amplitude of the increase in [Ca2+]i are the same in both regions, but it is clear that the Ca2+ signal initiates in the cell process. Solid bar indicates the time of application of 10 µM CCh.

As previously described (Larsson et al. 1998) individual cells differed in amplitude, rate of rise, and latency time of the Ca2+ response. Differences were not only detected among cells but also within a single cell. In cells with well-developed cellular processes, it was consistently observed that the CCh response initiated in the process and then took the form of a whole cell Ca2+ wave (Fig. 1C). In cells without well-defined cell processes, the Ca2+ response apparently started at the cell surface and then propagated toward the center of the soma. With 10 µM CCh, the amplitude, rate of rise, and decay of the Ca2+ response were much alike in all parts of the cell. This clearly suggests that the Ca2+ wave reflects regenerative Ca2+ release rather than Ca2+ diffusion from an active region. The speed of wave propagation amounted to 89 ± 8 µm s-1 (mean ± SE, n = 13). But in many instances the cell size was too small to accurately resolve the speed of propagation.

Ca2+ Responses at low agonist concentration

With 10 µM CCh the concentration of IP3 in the cell is likely to be supramaximal for Ca2+ release at all sites (EC50 for CCh is ~38 µM) (Larsson et al. 1998). With lower agonist concentrations the level of IP3 would only be sufficiently high at production sites, and therefore it was expected that regional differences in Ca2+ responses would be more pronounced. With CCh concentrations <1 µM, only a small proportion (36%) of the cells responded. Below 250 nM CCh, cells failed to respond.

With low agonist concentrations (500 nM) Ca2+ responses fell into two main categories: whole cell Ca2+ waves and Ca2+ responses with a different spatial profile (Fig. 2). In the latter group Ca2+ changes in cell processes appeared homogeneous or were confined to hot spots.



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Fig. 2. Different Ca2+ responses evoked by low concentrations of CCh. Ca2+ signals evoked by brief (5 s) pulses of low CCh concentrations (500 nM) appeared either as a whole cell Ca2+ wave or was primarily restricted to cell processes. In the latter, Ca2+ in the process was either homogeneously distributed or confined to hot spots. Data collected from 26 cells.

A number of cells (19% of the responding cells) displayed a whole cell Ca2+ wave with properties quite similar to Ca2+ waves induced by 10 µM CCh; i.e., the wave initiated in the process, propagated to the soma, but kinetics and amplitude were much alike in all parts of the cell (Fig. 3). The speed of wave propagation was, however, much lower than with high agonist concentrations and amounted to 36 ± 5 µm s-1 (n = 15).



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Fig. 3. Whole cell Ca2+ wave evoked by low concentrations of CCh. Top: pseudocolored Ca2+ images of a SH-SY5Y cell exposed to 500 nM CCh at times indicated by 1-4 in bottom panel. Line scan plot (top right) represents Ca2+ changes along the red line (A-B-C) depicted in image 4. The calculated wave velocity amounted to 35 µm s-1. Changes in [Ca2+] are expressed as %Delta F/F as indicated by the pseudocolor calibration bar 0-300%. Bottom: time course of Ca2+ signals in 2 selected regions. Traces were obtained by averaging %Delta F/F in a small region along the line scan as indicated by B (somatic region) and C (process region). Solid bar indicates the time of CCh application. Ryanodine (30 µM) was present.

In most responding cells (~80%), however, the Ca2+ profile displayed marked regional differences. The essential properties of these Ca2+ signals are illustrated in Figs. 4 and 5. Figure 4 shows a series of confocal images of a neuroblastoma cell together with the time course of [Ca2+] changes at three different cellular regions. Before stimulation, intracellular [Ca2+] was uniformly distributed throughout the cell. When the cell was stimulated with 500 nM CCh, a clear and fast Ca2+ transient was produced in the cell processes (positions A and C). This Ca2+ transient reached a peak level after ~0.5 s and then decayed to basal levels in different steps: after an initial fast decay, [Ca2+] leveled off or even slightly increased and finally slowly returned to resting levels. Despite CCh wash out for ~12 s, a second Ca2+ transient initiated at that time in both cellular processes. In the main cell body (position B), the CCh-induced Ca2+ signal had a complete different temporal profile. The increase in somatic [Ca2+] clearly showed two humps: a slow rise coinciding with the fast relaxing phase of the Ca2+ transient in the process was followed by a secondary slow rise of [Ca2+]. The second hump coincided with the [Ca2+] plateau in the processes. The decay of the somatic Ca2+ signal was apparently monophasic. So it is remarkable that within a single cell CCh can evoke Ca2+ signals with a completely different spatiotemporal domain.



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Fig. 4. Spatiotemporal properties of Ca2+ signals evoked by low concentrations of CCh: cell body vs. cell processes. Top panel: confocal images of an SH-SY5Y cell before and at the indicated times after stimulation with 500 nM CCh. Changes in [Ca2+] are expressed as %Delta F/F as indicated by the pseudocolor calibration bar 0-500%. The scale bar is 10 µm. Bottom panel: comparison of the time course of the Ca2+ signals at cell processes (red trace, ROI A; blue trace, ROI C) and cell body (green trace, ROI B). Note the occurrence of a secondary Ca2+ release event in cell processes. Solid bar indicates time of CCh application.



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Fig. 5. Spatiotemporal properties of Ca2+ signals evoked by low concentrations of CCh: homogeneous distribution vs. hot spot. A and B illustrate 2 cells exposed to 500 nM CCh for 5 s. Surface and contour plots represent Ca2+ signals along a line encompassing the process and the soma (indicated by a colored line in contour image of the cell). A: the [Ca2+] rise was seen simultaneously along the entire process. The Ca2+ signal in the soma was much smaller and delayed. Solid bar indicates the time of CCh application. B: the CCh-induced Ca2+ rise, measured at the peak of the response, was confined to a small region or hot spot in the cell process. Note different time scale in A and B.

Figure 5 shows two other examples and emphasizes that Ca2+ changes in the cell process either displayed a homogeneous spatial distribution or appeared in hot spots. Both cells were stimulated with 500 nM CCh for 5 s. The surface plots represent the Ca2+ response along the cell process up to the central part of the cell body. As observed in most cells, the CCh-induced Ca2+ increase in the process was not confined to one particular region or spot but rather extended over the whole distance of ~20 µm (plot A). The contour plot further indicates a simultaneous Ca2+ release throughout this structure. At the point where the cell process merged into the cell body, the signal was markedly delayed, and its amplitude rapidly decreased with distance. The other cell in Fig. 5 (plot B) demonstrates that in some cells the CCh-induced Ca2+ change was restricted to a small region in the cell process. These localized events or hot spots dissipated over a very short distance. At 50% of Ca2+ decay the average dimensions amounted to 5.5 ± 0.7 µm (n = 5). In general, the time course of localized and homogeneous Ca2+ transients in the process were quite similar. Hot spots were never observed in somatic regions. Finally, the size of the CCh-induced Ca2+ transients in the process showed marked variability irrespective of their spatial appearance. Figure 6 displays recordings of 17 cells exposed to low [CCh] (1 µM, 800 or 500 nM). Peak amplitudes of Ca2+ transients in processes varied between 20 and 500% Delta F/F. In most cells a single clear Ca2+ peak appeared in the process. In a number of cells (6 of 17), however, the Ca2+ transient in the process was preceded by one or more smaller Ca2+ peaks. In these cells the Ca2+ transient appeared along the entire process simultaneously, and the smaller peaks had a similar spatial distribution.



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Fig. 6. Different patterns of CCh-evoked Ca2+ signals in cell processes. Examples of Ca2+ responses during application of low concentration of CCh in cell processes of different cells. Different colors correspond to different [CCh] (500 nM red traces, 800 nM green trace, and 1 µM blue traces). Arrows indicate the occurrence of small events preceding a larger Ca2+ release signal.


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Spatiotemporal dynamics of Ca2+ signals were investigated in single neuroblastoma SH-SY5Y cells by fast laser-scanning confocal imaging.

IP3-induced Ca2+ release signals

As previously demonstrated (Larsson et al. 1998), stimulation of muscarinic receptors with CCh induced a rapid increase in intracellular Ca2+. This Ca2+ increase mainly reflects Ca2+ release from IP3-sensitive pools, because omitting extracellular Ca2+ or pretreatment of the cells with ryanodine had no obvious effects on the rise in intracellular [Ca2+]. In addition, almost all cells were sensitive to 10 µM CCh, but only a small proportion to 10 mM caffeine. This may indicate that the expression level of ryanodine receptors in neuroblastoma cells is quite low and varies greatly from cell to cell. Alternatively, the loading state of the caffeine-sensitive store may depend on cell activity, whereas that of the IP3-sensitive store would largely rely on a capacitive Ca2+ entry pathway.

Signals evoked by a high CCh concentration

In agreement with previous reports (Lambert and Nahorski 1990; Larsson et al. 1998; Murphy et al. 1991), 10 µM CCh evoked a single large Ca2+ transient in essentially all cells. Within an individual cell the profile of the Ca2+ transient was much the same in all regions of the cell, but it was also apparent that the signal initiated in cell processes before invading somatic regions. The wave speed was ~89 µm s-1, which is close to the value reported for bradykinin-induced Ca2+ waves in PC12 cells (Reber and Schindelholz 1996). The uniform rate of rise and amplitude of the Ca2+ response suggests that the wave is self-propagating (Clapham and Sneyd 1995). Because the caffeine-sensitive store appears not to be operative under our experimental conditions, we assume that wave propagation entirely relies on IP3Rs. It is evident that in cell processes, because of their small volume (or higher surface-to-volume ratio), [IP3] will raise much faster to threshold levels, and thus the latency before IP3-mediated Ca2+ release onset will be much shorter compared with the soma.

Signals evoked by low CCh concentrations

As expected, lower CCh concentrations (<= 1 µM) produced more restricted Ca2+ signals. Two distinct patterns of Ca2+ responses were observed: whole cell Ca2+ waves and spatially heterogeneous Ca2+ signals.

Whole cell Ca2+ waves had similar properties to those induced with 10 µM CCh, with the exception that the wave speed was markedly decreased (from 89 to 36 µm s-1). This finding suggests that under our experimental conditions diffusion of IP3 may limit the speed of wave propagation.

Most cells did not display a Ca2+ wave on stimulation with a low concentration of CCh. Instead, the CCh-induced Ca2+ signal showed marked spatial variability.

[Ca2+] changes were most prominent in cell processes and dissipated beyond the point where the cell process merged with the soma. In the cell soma a slow component was sometimes followed by a second, delayed component. The initial somatic component coincided with the decay of the Ca2+ signal in the process and thus mainly reflects diffusion of Ca2+ from the cell process into the cell soma (Eilers et al. 1995). The second delayed somatic component was sometimes obviously (2nd hump in Fig. 4) outweighing the Ca2+ level in the process. This resulted in Ca2+ diffusing back to the adjacent processes, producing an elevated plateau in the process. The second somatic component most likely reflects IP3-mediated Ca2+ release in the soma, the threshold of which can be set by the level of Ca2+ reached by diffusion from the cell process. Because the cell soma has a lower surface-to-volume ratio than the process, CCh will produce lower levels of IP3. Properties of Ca2+ signals are very sensitive to the IP3 concentration (Finch et al. 1991; Khodakhah and Ogden 1995; Parker et al. 1996), and therefore somatic Ca2+ release signals have a much smaller amplitude, a longer latency, and a slower rise time. In many instances the somatic Ca2+ release signal was not visible as a distinct peak but only produced an elevated somatic plateau. The ability of CCh to release Ca2+ in a cell compartment is thus essentially related to the rise in [IP3], and this will be largely determined by the surface-to-volume ratio of that compartment. For example, the surface-to-volume ratio of a cell process with a radius of 1.5 µm and a length of 25 µm would amount to 1.42, as compared with 0.73 for a cell soma with a radius of 6 µm and a height of 5 µm. Therefore IP3 levels reached in the cell soma would only be about one-half of those obtained in the cell process.

With low CCh concentrations, Ca2+ signals in cell processes initiated simultaneously along the entire process, termed homogeneous Ca2+ signal, or were sometimes restricted to a small area, termed "hot spot." The diameter of hot spots was ~5.5 µm, allowing them to be identified as elementary Ca2+ release signals or Ca2+ puffs described in other cell types (Bootman et al. 1997; Koizumi et al. 1999; Yao et al. 1995). No gross differences were seen between the kinetics and amplitude of hot spots and homogeneous Ca2+ signals. Therefore it is very conceivable that homogeneous Ca2+ signals arise from the simultaneous activation of a small number of elementary Ca2+ release sites. The finding that the homogeneous Ca2+ release signal was composed of multiple peaks with a diameter of ~5 µm (Fig. 5B) supports this idea. Homogeneous Ca2+ signals were more frequently observed than hot spots. This indicates that IP3Rs are distributed along the whole length of the process and suggests that the distance between release sites is too short to allow activation of a single site.

In PC12 and hippocampal neuronal cells, Ca2+ puffs occur repetitively in the maintained presence of threshold concentrations of IP3-generating agonists (Koizumi et al. 1999; Reber and Schindelholz 1996). With prolonged threshold CCh stimulation, we also observed repetitive Ca2+ puffs in SH-SY5Y cells (data not shown). These Ca2+ puffs could be observed in both the cell body and the processes. The reason for the difference between brief and prolonged application of agonist may be related to the IP3 gradient. With brief CCh application, the difference in surface-to-volume ratio will create a large IP3 gradient between the cell process and cell soma. With prolonged CCh application, this IP3 gradient will dissipate resulting in a threshold elevation of IP3 throughout the whole cell.

Conclusions

To convey information to other cells, neurons have developed complex regulating mechanisms, many of which depend on changes in intracellular [Ca2+]. Yet, the present experiments suggest that a change in the surface-to-volume ratio is a very simple way for a neuronal cell to alter the spatiotemporal domain of its Ca2+ response and thus to determine the intracellular messenger function of Ca2+.


    ACKNOWLEDGMENTS

J. B. Parys is Research Associate and P. De Smet is Senior Research Assistant at the Foundation for Scientific Research---Flanders. G. Bultynck is Predoctoral Fellow at the "Vlaams Instituut voor de bevordering van het Wetenschappelijk-Technologisch Onderzoek in de industrie."

This work was supported by Grants 3.0238.95, 3.0207.99, and G.0186.97 from the Foundation for Scientific Research---Flanders and by Grants 94/04 and 99/08 of the Concerted Actions K. U. Leuven.


    FOOTNOTES

Address for reprint requests: G. Callewaert, Laboratory of Physiology, K. U. Leuven, Campus Gasthuisberg, 3000 Leuven, Belgium.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 July 1999; accepted in final form 19 October 1999.


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