Two Mechanisms That Raise Free Intracellular Calcium in Rat Hippocampal Neurons During Hypoosmotic and Low NaCl Treatment

Aren J. Borgdorff,1 George G. Somjen,2 and Wytse J. Wadman1

 1Institute for Neurobiology, University of Amsterdam, 1098 SM Amsterdam, The Netherlands; and  2Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710


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ABSTRACT
INTRODUCTION
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Borgdorff, Aren J., George G. Somjen, and Wytse J. Wadman. Two Mechanisms That Raise Free Intracellular Calcium in Rat Hippocampal Neurons During Hypoosmotic and Low NaCl Treatment. J. Neurophysiol. 83: 81-89, 2000. Previous studies have shown that exposing hippocampal slices to low osmolarity (pi o) or to low extracellular NaCl concentration ([NaCl]o) enhances synaptic transmission and also causes interstitial calcium ([Ca2+]o) to decrease. Reduction of [Ca2+]o suggests cellular uptake and could explain the potentiation of synaptic transmission. We measured intracellular calcium activity ([Ca2+]i) using fluorescent indicator dyes. In CA1 hippocampal pyramidal neurons in tissue slices, lowering pi o by ~70 mOsm caused "resting" [Ca2+]i as well as synaptically or directly stimulated transient increases of calcium activity (Delta [Ca2+]i) to transiently decrease and then to increase. In dissociated cells, lowering pi o by ~70 mOsm caused [Ca2+]i to almost double on average from 83 to 155 nM. The increase of [Ca2+]i was not significantly correlated with hypotonic cell swelling. Isoosmotic (mannitol- or sucrose-substituted) lowering of [NaCl]o, which did not cause cell swelling, also raised [Ca2+]i. Substituting NaCl with choline-Cl or Na-methyl-sulfate did not affect [Ca2+]i. In neurons bathed in calcium-free medium, lowering pi o caused a milder increase of [Ca2+]i, which was correlated with cell swelling, but in the absence of external Ca2+, isotonic lowering of [NaCl]o triggered only a brief, transient response. We conclude that decrease of extracellular ionic strength (i.e., in both low pi o and low [NaCl]o) causes a net influx of Ca2+ from the extracellular medium whereas cell swelling, or the increase in membrane tension, is a signal for the release of Ca2+ from intracellular stores.


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Acute hemodilution, whether caused by overhydration or by salt loss, causes neurological symptoms (Arieff and Guisado 1976; Avner 1995). Lowering the osmolarity (pi o) of a solution bathing brain slice preparations lowers the threshold for the induction of epileptiform discharges (Andrew 1991; Andrew et al. 1989; Dudek et al. 1990; Roper et al. 1992), promotes burst firing (Azouz et al. 1997), and in extreme cases causes recurrent episodes of spreading depression (Chebabo et al. 1995a). Excitatory synaptic transmission is enhanced in low and depressed in high pi o (Chebabo et al. 1995a; Huang et al. 1997; Huang and Somjen 1995; Rosen and Andrew 1990). Low pi o also causes a striking reversible and concentration-dependent lowering of interstitial calcium ([Ca2+]o) in hippocampal tissue slices (Chebabo et al. 1995b). Lowering extracellular NaCl concentration ([NaCl]o) while keeping osmolarity constant by substituting mannitol or fructose also enhances synaptic transmission, albeit less powerfully than low pi o (Chebabo et al. 1995a; Huang et al. 1997). It also depresses [Ca2+]o (Chebabo et al. 1995b). Stabel et al. (1990) reported depression of orthodromic evoked potentials when Na+ was replaced by impermeant large cations. It seems that replacing electrolytes with an uncharged compound has an effect that is different from changing a permeant to an impermeant ion.

From these earlier observations, we concluded that lowering [NaCl]o is in itself a potentiator of synaptic transmission. Because the effect in synapses was consistently greater in low pi o than in isoosmotic low [NaCl]o solution, it seemed that hypotonia-induced dendritic swelling added to the potentiation. We suggested, hypothetically, that the fraction of the potentiation that could be attributed to the low [NaCl]o is caused by the uptake of Ca2+ into presynaptic terminals and also into postsynaptic elements (Chebabo et al. 1995a). Impaired membrane Na-Ca countertransport caused by lowering extracellular sodium concentration ([Na+]o) (Eisner and Lederer 1985) seemed to be a plausible explanation for the accumulation of Ca2+ in neurons. To test some of these hypothetical proposals, we measured changes of [Ca2+]i in hippocampal neurons exposed to low pi o or to low [NaCl]o, both in the presence and in the absence of external Ca2+. We also examined whether substituting choline+ for Na+ or methyl-sulfate- for Cl- would have the same effect as substituting mannitol for NaCl.

Some of the results were been reported in Somjen (1997, 1999a) and Somjen et al. (1997).


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The experiments were conducted at Duke University (confocal microscopy) and the University of Amsterdam (dual emission fluoroscopy). To prepare hippocampal tissue slices, rats of 80-120 g body weight were decapitated under ether anesthesia, their brains were removed, the hippocampi were dissected, and transversal slices of 250-300 µm were cut. Slices were maintained in a recording chamber on the platform of an inverted microscope (Nikon Diaphot). They were kept at a temperature of 32°C and perfused with a solution of (in mmol/l) NaCl 120, KCl 3.5, CaCl2 2.5, MgSO4 1.3, NaH2PO4 1.25, NaHCO3 25, D-glucose 10, pH 7.3, and gassed with 95% O2/5% CO2. Extracellular pi o was lowered by deleting NaCl, and in isoosmotic low NaCl medium, mannitol replaced NaCl. Sharp electrodes (impedance >80 MOmega ) filled with 3 M K-acetate, to which 20 mM fura-2 potassium salt was added for calcium recordings, were used to penetrate CA1 pyramidal cells. Membrane voltage was recorded with a bridge amplifier (IR283, Neurodata). For calcium measurements, a 20× objective 0.7 N. A. Fluor Nikon was used in combination with a custom-made excitation wavelength switcher that excited at 340 or 380 nm from an Hg-bulb source. Fluorescence was measured from the soma region with a cooled integrating CCD camera (CE200, Photometrics) that was synchronized with the electrophysiological protocols. Data were corrected off-line for background and fluorescence ratios were determined. Absolute calibration of calcium concentration was not attempted in the slice preparation.

CA1 pyramidal neurons were isolated according to the method of Kay and Wong (1986). Briefly, Wistar rats of 80-150 g body weight were used and hippocampal slices of 400-500 µm were cut as described in the preceding paragraph. The CA1 region was cut into smaller fragments of ~0.4 × 0.4 mm. These were digested at either 32°C for 45-60 min or at room temperature for 75 min. The digestion medium contained (in mmol/l) NaCl 125, KCl 5, CaCl2 1, MgCl2 2, D-glucose 25, [2-hydroxyethyl]piperazine-[2-ethanesulfonic acid] 10, pH 7.0, with trypsin 1.0 or 0.75 mg/ml. The digestion medium was kept under oxygen atmosphere and stirred gently. After digestion, the tissue pieces were washed three times and then maintained in trypsin-free oxygenated medium at room temperature. Tissue fragments were dispersed by trituration with a graded series of fire-polished Pasteur pipettes. Cells were allowed to settle in a chamber on the microscope stage and were then maintained in flowing medium of the following composition (in mmol/l): NaCl 133, KCl 3.5, CaCl2 1.2, MgCl2 1.0, glucose 25, HEPES 10, pH 7.3, at 22-26°C. There was no need to coat the chamber floor with adhesive because the cells remained in place in the steadily flowing solution. Spindle-shaped or pyramidal-shaped "shiny" well-filled cells with "smooth" cytoplasm and with a well-shaped (sometimes forked) apical dendritic stump of at least 2 soma lengths, occasionally with small basal dendrite remnants, were selected for recording. Very probably all were CA1 pyramidal neurons. Extracellular pi o or NaCl was lowered as for slices except that occasionally sucrose or fructose was used instead of mannitol for isoosmotic NaCl replacement. Average osmolarities (as measured by freezing point osmometry) were as follows: normal solution, 301 mosm/kg; moderately hypoosmolar solution with 40 mM NaCl deleted, 231 mosm/kg; severely hypoosmolar solution with 60 mM NaCl deleted, 204 mosm/kg.

For calcium measurement, the tissue fragments were loaded before trituration for 5 min at 30°C with 2 µM fura-2 AM fluorescent indicator dye. The cells were allowed to hydrolyze the fura-2 AM for >= 15 min before recording started. Calibration was performed on isolated neurons under calcium-free (EGTA) and saturated (ionomycin) conditions. A Kd of 220 nM for fura-2 was used for the conversion of fluorescence ratio to calcium concentration. Fluorescence was recorded with the same optical and computer equipment as for the slice experiments except that occasionally a 40× objective (Fluor 1.3 N. A., Nikon) was used.

Confocal microscopy was used for the simultaneous determination of cell size and [Ca2+]i changes. Cells from hippocampal tissue fragments were dispersed by trituration into HEPES-buffered bathing solution as described in the preceding two paragraphs, except that the solution contained a mixture of the membrane-permeant AM esters of the fluorescent dyes fluo-3 (2 µM) and fura-red (10 µM). After 10 min of incubation, the suspension was placed in the recording chamber and, after allowing an additional 7 min for the cells to settle to the bottom, perfusion of the chamber with dye-free HEPES-buffered solution began. An image of one or two cells was recorded with a 40× water immersion objective of a Zeiss Axioskop microscope and a BioRad MRC-600 confocal imaging system with COMOS software. Fluorescence was recorded at 10 or 20 s intervals from one or two selected areas in the image of each cell at 488 nm wavelength of excitation, and at the 520 and 640 nm emission wavelengths of, respectively, fluo-3 and fura-red. Images were stored at 30 or 60 s intervals. Changes in [Ca2+]i were determined from changes of the ratio of fluorescences at 520/640 nm. Fluo-3 fluorescence intensity at 520 nm increases whereas that of fura-red at 640 nm decreases when [Ca2+]i increases (Haugland 1996). Ratios were computed off-line after subtraction of "background." The image areas of cells were measured off-line by drawing the outline of a cell's image and reading the area in µm2 with the aid of the COMOS software. Volumes were computed as the 3/2 power of image area and their changes were expressed as percent of control volume.

Data are given as means ± SE. Comparisons were performed with Student's t-test. Correlation was calculated with linear regression. P < 0.05 was considered to indicate a significant difference.


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[Ca2+]i in freshly dissociated neurons

[Ca2+]i in isolated cells was measured in two different ways. Cells filled with a mixture of the dyes fluo-3 and fura-red were viewed in a confocal microscope, and size changes could be measured at the same time as the fluorescence emission ratio of the two dyes. However, quantitative calibration of the fluorescence ratio proved to be elusive. In another series of experiments, cells were filled with fura-2 and in these cases absolute calibration yielded reliable [Ca2+]i values. Of fura-2 stained cells, however, no images were recorded that allowed the estimation of cell size (see METHODS).

Figure 1 shows sample confocal images. Most cells were subjected to two trials: in some cells the effect of isoosmotic lowering of [NaCl]o was compared with that of lowering pi o (at equimolar [NaCl]o), whereas in other cells two different levels of pi o were compared. Exposures lasted 5-7 min. The order of presentation of the test solutions was varied among cells.



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Fig. 1. Fluorescence images of two dye-filled neurons seen in one optical field, excited at 488 nm, and recorded near the emission maxima of the dyes fluo-3 (520 nm) and fura-red (640 nm). C, control images just before changing solution. LNa, after 5 min exposure to mannitol-substituted isosmotic low NaCl solution. During low NaCl treatment, the fluo-3 fluorescence increased and the fura-red fluorescence decreased, indicating elevation of intracellular calcium activity ([Ca2+]i).

In the gray-scale reproductions in Fig. 1, the differences in fluorescence intensities are not striking but the brightening of the fluo-3 and the darkening of the fura-red images in the low NaCl compared with the control condition are apparent. The changes become obvious in Fig. 2, where the computed fluorescence ratios in the two cells are plotted against time. During the initial control period, the ratio gradually decreased and then, when [NaCl]o was lowered, it sharply increased in both cells. The ratio then recovered during washing and increased once more when pi o was lowered. The "baseline" fluorescence ratio under control conditions declined in some and increased in other cells, possibly in part due to changes in "resting" [Ca2+]i but possibly also because of unequal rates of decay in the fluorescence of the two dyes. Ratio changes caused by lowering pi o or [NaCl]o were superimposed on such baseline drifts and were always quite obvious from the change in the direction of the trace. After 5 min of administration of moderately hypoosmotic solution (pi o approximately -70 mosm/kg), the fluorescence ratio increased to 124 ± 6% of the value recorded immediately before changing solution (n = 12). The effect was extremely variable, with a range of 101-173%. With 60 mM NaCl deleted (pi o about -97 mosm/kg), the increase was 132 ± 12% (n = 14, range = 105-177%). As in our previous study (Aitken et al. 1998), the degree of cell swelling was also very variable. Figure 3 plots normalized fluorescence ratio against relative cell volume at the end of hypotonic trials of individual cells. Even though, as a group, the cells that swelled most also showed the greatest increase in fluorescence ratio, the correlation is not significant (R = 0.27). When 60 mM NaCl was substituted by equiosmolar mannitol or fructose, the cells did not detectably swell (final mean volume, 97%). The fluorescence ratio increased to a mean of 127 ± 5% (n = 12, range = 100-145%), which is not significantly different from the mean ratio measured during exposure to low pi o solution of the same [NaCl]o.



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Fig. 2. Fluorescence ratio changes in the two cells illustrated in Fig. 1. Normal bathing solution was replaced for 5.5 min first with mannitol-substituted isoosmotic low NaCl solution then, after washing with normal solution, for 5 min with hypoosmotic solution. "Baseline" fluorescence ratio appears to decrease during more than 30 min of recording. Note rapid, initial, apparent surge of [Ca2+]i followed by sustained elevation during low NaCl administration and delayed increase of [Ca2+]i during low pi o treatment. Fluorescence was measured at 20 s intervals.



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Fig. 3. Changes in fluorescence ratio plotted against changes in cell volume as percentage of preexposure control at the end of hypoosmotic treatment, measured in the presence of normal bath calcium. Cell volume was estimated from images similar to Fig. 1, as described in METHODS. Each point represents a different cell. The cells were filled with the dyes fluo-3 and fura-red. open circle , moderate hypotonicity, 40 mM NaCl deleted. , severe hypotonicity, 60 mM NaCl deleted. No significant correlation found (R = 0.27).

In the trials in which fura-2 was the indicator, each cell was exposed to mannitol-substituted low NaCl as well as to moderately low pi o (Delta [NaCl]o -40 mM), with the sequence of administration reversed in half the trials. Additional cells were used to test the effect of substituting choline-chloride for NaCl, and some of these cells were also exposed to low pi o (see under Effect of ion substitutions). The control level of [Ca2+]i in all these cells was 83 ± 7 nM (n = 18). At the end of 5 min exposure to hypotonic solution, [Ca2+]i increased to 155 ± 20 nM (n = 15, P < 0.0005, paired t-test) whereas mannitol-substituted isosmotic low NaCl treatment resulted in a mean of 101 ± 11 nM (n = 12, P < 0.01). After the treatments, washing with normal solution caused [Ca2+]i to return to 88 ± 8 nM.

[Ca2+]i changes in zero [Ca2+]o

To investigate whether the excess Ca2+ appearing in cytosol during low pi o and low [NaCl]o treatment is derived from the external solution or from intracellular stores, cells were loaded with the mixture of fluo-3 and fura-red dyes and then washed in calcium-free bathing solution (with 1 mM EGTA in the bath) for 5-8 min. Removing calcium from the bath lowered the fluorescence ratio in cells to 88 ± 2% of "normal" (n = 13). Cell volume did not change (99.7%).

Isoosmotic lowering of NaCl by 60 mM (mannitol or sucrose substitution) triggered an immediate but brief rise of fluorescence ratio followed by return to baseline level. In hypotonic solution, the fluorescence ratio increased if and when the cell swelled. As we described earlier (Aitken et al. 1998), cell swelling was frequently delayed by several minutes after changing the bath to hypotonic solution, and in these cases the increase in fluorescence ratio was similarly delayed (Fig. 4). After 6-9 min exposure to hypotonic solution (60 mM NaCl deleted, Delta pi o = -108 mosm/kg) in the absence of external calcium, the fluorescence ratio increased to 109 ± 2% (n = 17, range 97-122%) compared with the value measured in zero calcium, normal pi o. Hypotonic swelling of the same cells amounted to 117 ± 3% (n = 17, range 100-145%). In zero external calcium, the increase in fluorescence ratio was correlated with the degree of cell swelling (Fig. 5; R = 0.80, P < 0.001), which is clearly different from the more random scatter of the correlogram representing cells in normal [Ca2+]o (Fig. 3).



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Fig. 4. Fluorescence ratio (continuous line) and cell volume (open circle ) of an isolated neuron in the absence of external calcium. Low NaCl, 60 mM of NaCl deleted, substituted by mannitol. Hypoton., 60 mM NaCl deleted. Note brief rise of [Ca2+]i at onset of low NaCl administration followed by return to baseline contrasted with delayed but persistent elevation of the fluorescence ratio during hypotonicity, which parallels cell volume increase. Fluorescence was measured every 20 s, images were recorded at 60 s intervals.



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Fig. 5. The correlation of fluorescence ratio (fluo-3/fura-red) and cell volume in the absence of external calcium. Data taken from 17 cells at the end of hypoosmotic treatment (60 mM NaCl deleted), normalized to the last prehypotonic control value measured in zero calcium. In the absence of calcium in the bath, the correlation was significant (R = 0.80, P < 0.001).

Twelve of the 17 cells were also tested in zero [Ca2+]o isoosmotic low [NaCl]o solution, either before or after hypotonia. The transient "calcium spike" was seen whenever low [NaCl]o administration preceded low pi o, as in Fig. 4. However, by the end of 6-8 min in zero external calcium low [NaCl]o, the ratio was near control level (101 ± 5% of that measured in zero calcium normal [NaCl]o). The mean cell volume was 91 ± 3% at the end of low [NaCl]o treatment in zero [Ca2+]o.

Effect of ion substitutions

To investigate whether the lowering of Na+ by itself would also raise [Ca2+]i, four cells were filled with fura-2 AM and exposed to choline-Cl-substituted low [NaCl]o solution followed by lowering of pi o. Three additional cells were tested with choline-Cl-substituted solution only. The substitution of choline+ for Na+ did not cause an elevation of [Ca2+]i. In this sample, the resting [Ca2+]i was only 66 nM and it remained at 65 ± 6 nM (n = 7) during choline-Cl administration. After washing with normal solution it was 64 ± 8 nM. In four of these cells, subsequent hypotonia raised the fluorescence ratio as usual.

Because substituting Na+ with choline+ had no effect on [Ca2+]i, this left either Cl- or total ionic strength to account for the raising of [Ca2+]i by low [NaCl]o. To investigate the effect of Cl-, 12 cells were exposed to bathing solution in which 60 mM NaCl was replaced with Na-methyl-sulfate. Seven of these cells were also tested in mannitol- or sucrose-substituted low [NaCl]o and five were also tested in hypoosmolar solution. [Ca2+]i was estimated as the fluorescence emission ratio of fluo-3 and fura-red dyes. At the end of 7 min in Na-Me-sulfate, the fluorescence ratio was 101 ± 3% (n = 12, range 90-109%). In this sample, lowering [NaCl]o raised the ratio to 124 ± 8% (n = 7) and lowering pi o caused it to rise to 116 ± 7% (n = 5).

[Ca2+]i in pyramidal cells in hippocampal slices

Of 10 cells studied in hippocampal slices, four were allowed to fill with the indicator fura-2 and then the microelectrode was withdrawn from the cell. In these cells, the fluorescence ratio was recorded but electrophysiological recordings were not made. Two of these cells were left undisturbed and only "resting" fluorescence was registered. The other two cells were stimulated by orthodromic volleys. Such synaptically transmitted stimuli caused brief increases in [Ca2+]i, which registered as increase of the fluorescence ratio (Fig. 6). The other six cells were studied with the microelectrode remaining inserted. In these cells, the membrane potential was recorded as well as the electrical responses either to synaptic stimuli or to direct depolarizing pulses (Figs. 7 and 8).



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Fig. 6. The effect of lowering pi o on the fura-2 fluorescence excitation ratio in a neuron of a hippocampal slice. At 2-min intervals, the cell was stimulated by orthodromic volleys, causing the transient increases in fluorescence ratio. During hypoosmotic exposure (heavy horizontal line), baseline fluorescence ratio as well as its transient responses were initially depressed and then increased. Washing with normal solution brought partial recovery. After 8 min of washing, the fluorescence ratio suddenly increased much beyond the scale of this figure. Such explosive increases of [Ca2+]i usually signal the demise of the cell.



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Fig. 7. Orthodromically evoked membrane voltage and fluorescence ratio responses before and during hypoosmotic exposure of a neuron in a hippocampal slice. A: intracellular potential response in control condition. B: response recorded after 11.5 min of hypoosmotic treatment (40 mM NaCl omitted), superimposed on the control response from A. C: response after 17 min of hypoosmotic exposure, superimposed on the same control; note slight depolarization. Note also broadening of the excitatory postsynaptic potential (EPSP) and multiple firing during hypotonia. D: fura-2 fluorescence ratio responses associated with the electric responses of A-C; zero-time is the moment of stimulation. The "baseline" fluorescence ratio had increased, but for this illustration the responses were shifted to a common initial level to emphasize the increase in stimulus-evoked responses.



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Fig. 8. Membrane voltage responses (A and B) and the corresponding transient fluorescence ratio responses (C) evoked by depolarizing current pulses recorded in normal artificial cerebrospinal fluid (ACSF) and in low pi o (40 mM NaCl omitted) of a neuron in a hippocampal slice. B: note change in firing pattern and appearance of a slow depolarizing wave on which the first five spikes are riding. Membrane potential was initially -65 mV, but during hypotonia it first hyperpolarized to -73 mV and then returned to approximately -64 mV. During washing (not shown), the cell depolarized to -60 mV.

The dye concentration, estimated as the weighted mean of the fluorescence intensities at the two excitation wavelengths, tended to decrease in cells from which the electrode was withdrawn, suggesting leakage or bleaching. In the cells with electrode remaining inserted, the mean fluorescence tended to increase, indicating continuing diffusion from pipette into cell. The "resting" fluorescence ratio was, however, more stable than the absolute florescence intensities.

Slices were exposed to moderately low pi o (NaCl reduced by 40 mM; pi o reduced by ~70 mosm/kg) for 15-22 min. During hypotonic exposure in 9 of the 10 cells, the "resting" or "baseline" fluorescence ratio decreased transiently and then it increased. In 9 of the 10 cells, the final value was above the initial control level. The minimum was reached in 3-12 min, the rapidity of the effect probably being influenced mainly by the distance of the cell from the slice surface, which determines the equilibration with the bath. During washing with normal solution, the fluorescence ratio returned partially or completely to its control level. At its minimum, the "resting" fluorescence ratio decreased to 94 ± 1.2% of control and at the end of hypotonic treatment it rose to 106 ± 2% (n = 10, P < 0.035, paired t-test). At the end of the recovery period it returned to 100 ± 3%.

During hypotonic exposure, the transient fluorescence ratio responses evoked either by DC pulses or by synaptic stimulation were initially depressed in four of eight cells and were then enhanced in six of eight cells. When the initial depression was observed, the stimulus-evoked responses changed pari passu with the baseline level (Fig. 6). At the end of the hypotonic treatment, the mean of the maximal evoked responses of all cells was 107% of control, whereas for the six cells that showed an increase the maximum was 152 ± 21% returning, after washing with normal solution, to 112%. Within the resolution of the recordings, there was no difference in the time constant of the recovery of the calcium signals for the various conditions. Excitatory postsynaptic potentials (EPSPs) were recorded in two cells. In both, the EPSP increased, which confirmed the report by Huang et al. (1997). In the examples shown in Fig. 7, the enhancement was quite pronounced, which resulted in multiple firing. These potential responses were reminiscent of paroxysmal depolarization (PDS; see, e.g., Wong and Prince 1979). There was a corresponding increase in the stimulus-evoked Delta [Ca2+]i (Fig. 7D).

Four cells were stimulated by injection of depolarizing current pulses of varying strengths (0.05-0.45 nA). In one of these cells, strong pulses triggered a depolarizing slow wave when pi o was lowered. This slow wave was similar to the ones observed by Huang et al. (1997) (see DISCUSSION). The fluorescence ratio transients were markedly potentiated (Fig. 8). When stimulus-evoked Delta [Ca2+]o transients were plotted against the number of action potentials fired, the average Delta [Ca2+]o per spike increased to 122% of control at the end of hypotonia (n = 5). This disproportionate increase may be attributed in part to the appearance of putative calcium currents as in Figs. 7 and 8, but it was also observed in the absence of evidence of an increased calcium current. An additional factor may be an increase in action potential duration (Figs. 7 and 8). During the administration of hypoosmotic fluid, the resting intracellular potential changed only slightly and seemingly at random, sometimes hyperpolarizing, sometimes depolarizing or not changing (e.g., Fig. 7, B and C). The input resistance was measured with small hyperpolarizing current pulses. In low pi o it increased in all 5 neurons measured, from a mean control value of 108 ± 26 to 140 ± 31 MOmega . Next it returned to 109 MOmega in agreement with previous observations on neurons in slices as well as in isolation (Huang et al. 1997; Somjen et al. 1993).


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lowering pi o or [NaCl]o raises [Ca2+]i in neurons

In both preparations, tissue slices and dispersed cells, exposure to hypoosmolar solution caused [Ca2+]i to increase, but in cells within the slice the rise was usually preceded by a transient decrease. In the cells in tissue slices, the average final increase in [Ca2+]i seemed milder than in dispersed cells. It could be that under the conditions in the slice, two opposing processes were at work: one tending to depress and the other to raise [Ca2+]i, with the former dominating at the start of hypotonia and the latter gaining the upper hand later. Among the differences between the two preparations, slices and dispersed cells, are the slow equilibration of interstitial fluid with the bathing fluid (Chebabo et al. 1995b) and the higher temperature of slice preparations. Both factors could favor compensatory regulation of [Ca2+]i. Moreover, in bicarbonate buffered solutions, intracellular pH is better regulated than in HEPES buffered solutions (Bevensee et al. 1996), and pH influences the active fraction of Ca2+ (Tombaugh and Somjen 1998).

The effect of low pi o on [Ca2+]i appeared to be similar regardless of the indicator dyes that were used. On the other hand, isoosmotic lowering of [NaCl]o seemed to have the same effect as lowering pi o when the mixture of fluo-3 and fura-red was used, but low [NaCl]o had a weaker effect in the trials using fura-2. The discrepancy could mean that the stains distribute differently among cellular compartments. Synaptic transmission is augmented more powerfully by hypotonia compared with low [NaCl]o (Chebabo et al. 1995a; Huang et al. 1997), which could be caused by a greater accumulation of Ca2+ in presynaptic terminals under the influence of low pi o.

In an earlier study, we found that interstitial calcium concentration in hippocampal slices decreased when hippocampal slices were exposed either to low pi o or to isosmotic low [NaCl]o (Chebabo et al. 1995b). The Ca2+ that disappeared from the extracellular fluid presumably entered cells, but it is not clear how much was taken up by glial cells and how much by neurons. Nor do extracellular measurements reveal the fraction that remains free relative to what is sequestered or bound inside cells. A simple calculation suggests that only a small fraction can remain active in solution. When [NaCl]o was lowered by 40 mM either isotonically or hypotonically, [Ca2+]o decreased ~300 µM (Chebabo et al. 1995b). Assuming that swollen cells occupied 90% of tissue volume, the 300 µM removed from extracellular fluid would be diluted to about 33 µM. Even though we have fura-2 fluorescence ratio calibrations for dispersed cells only, the changes in excitation ratios of the cells in the slices indicate that the increase in free [Ca2+]i must have remained well below 1 µM, more than an order of magnitude less than the calculated number. However, this increase is to be expected if the calcium buffer capacity of CA1 neurons caused by fast binding and sequestration is on the order of 100-200, which is the value estimated for these neurons (Wadman and Borgdorff 1999). In addition, disproportionate calcium uptake by glial cells could contribute.

Lowering [NaCl]o stimulates uptake of Ca2+ from the extracellular medium whereas hypotonia triggers release from intracellular stores

In the presence of normal external Ca2+, the degree of cell swelling had only a weak relationship to the magnitude of the increase of [Ca2+]i (Fig. 3), and isotonic lowering of [NaCl]o caused a comparable if sometimes weaker effect with equivalent lowering of pi o. When external calcium was removed, lowering pi o still caused elevation of [Ca2+]i albeit less than in normal calcium, but lowering of [NaCl]o now triggered only a short-lived increase that dissipated in a few minutes. Moreover, in the absence of external calcium, there was a strong correlation between the degree of cell swelling and the final level to which [Ca2+]i rose during hypotonia (Fig. 5). It seems that two independent mechanisms were in operation. Lowering [NaCl]o caused the uptake of Ca2+ from the external medium whereas hypotonic swelling triggered the release of Ca2+ ions from intracellular stores. Release of Ca2+ from intracellular stores triggered by hypotonicity has also been reported for cell types other than neurons (Missiaen et al. 1997, 1998). Low pi o in the presence of normal external Ca2+ appears to induce both influx from the extracellular medium and release from intracellular stores. The stimulus that initiates the swelling-induced calcium release could be either the osmotic influx of water or the tension of the stretched plasma membrane. Lowering the concentration of metabolic substrates, ATP, or catalysts in cytosol is probably of minor importance because cell volume can expand only by a fraction of its normal size (Chebabo et al. 1995a). Mediation by some intracellular messenger signal, for example inositol trisphosphate, is certainly possible. Also, hypotonic swelling of organelles, particularly endoplasmic reticulum and mitochondria, could induce the release of stored calcium. The data do not permit a choice among these and perhaps other mechanisms.

The rapid calcium transient seen at the onset of low [NaCl]o administration in zero calcium (Fig. 4) must also represent release from internal stores. Such a transient release seems also to occur in normal external calcium in addition to the uptake from the outside, as seen from the brief extra boost of fluoresence ratio at the onset of low NaCl treatment in both cells represented in Fig. 2.

Uptake of Ca2+ is not mediated by the Na-Ca exchanger, nor does it depend on generalized membrane ion "leak"

The change in [Ca2+]i was not directly related to water uptake because dilution would have lowered, not raised, the concentration. Moreover, the relative high buffer capacity of these neurons would counteract most of the volume- induced changes. Also, the effect is not caused by change in membrane potential or of resting membrane resistance ("leak"). Neither in this series, nor in our earlier experiments, did the "resting" membrane properties change in a way that could explain the change in [Ca2+]i (Huang et al. 1997; Somjen et al. 1993). Linear "leak" conductance usually decreased in both low [NaCl]o and low pi o. This does not exclude a net increase in the inward flux of calcium ions because the resting calcium conductance is a small fraction of the total membrane conductance, but it does exclude a nonspecific generalized increase in ion conductance.

In our previous reports, we hypothetically attributed the elevation of [Ca2+]i to the suppression or reversal of the Na/Ca exchanger by low [Na+]o (Chebabo et al. 1995b; Huang et al. 1997). This suggestion is now refuted because substituting choline+ for Na+ failed to raise [Ca2+]i. Nor does the effect depend on low Cl- concentration becuase substituting Me-sulfate- for Cl- had no effect on [Ca2+]i either. We must conclude that the uptake of Ca2+ from the medium was triggered by the low total ionic strength of the medium. Substitution by choline+ in this study may be compared with Stabel et al.'s (1990) use of Tris to replace Na+. In their experiments, orthodromically evoked responses were depressed presumably because the driving force for Na+ currents was reduced. In our trials, when NaCl was replaced by mannitol or fructose, extracellular synaptic potentials as well as whole-cell recorded synaptic currents were greatly enhanced (Chebabo et al. 1995a; Huang et al. 1997). It appears that the potentiating effect of the reduced extracellular ionic strength more than offsets the depressant effect of the diminished Na+ concentration gradient. Replacing Na+ with an impermeant cation such as choline or Tris does not potentiate Ca2+ influx.

The use of different preparations and different experimental conditions

Isolated cells are more accessible to optical recordings, their environment equilibrates with changing bath solutions much faster, and they generally lend themselves more readily to experimental manipulation. Cells within tissue slices retain their natural environment. The warmer temperature at which interfaced slices are maintained and the presence of CO2 and bicarbonate resemble physiological conditions. Thus the advantages and disadvantages of isolated cells and tissue slice preparations are complementary. Similarity of the outcome reinforces the validity of experiments repeated under different conditions. The trials described in this paper were conducted at two different institutes using several different experimental setups. Bathing solutions, temperatures, and oxygenation were not exactly alike, yet the results were similar.

The calcium concentration of the bath for slices was 2.5 mM. This is higher than the 1-1.5 mM found in normal cerebrospinal fluid of mammals (Katzman and Pappius 1973), but it is "traditional" for slice preparations (DiScenna 1987). It should be noted that in our previous studies on the effects of osmolarity, hippocampal slices were bathed in artificial cerebrospinal fluid (ACSF) containing only 1.2 mM calcium (Chebabo et al. 1995a,b; Huang et al. 1997). The bicarbonate in ACSF chelates a fraction of the calcium, as does the bicarbonate of cerebrospinal fluid and extracellular fluid in live brains (Schaer 1974; Somjen et al. 1987). The solutions used for the dissociated cells in confocal microscopy had only 1.2 mM calcium and these were buffered by HEPES, which does not bind calcium.

Role of voltage gated calcium channels

For the modulation of synaptic transmission, stimulus-induced Delta [Ca2+]i responses are even more important than is baseline [Ca2+]i. The number of intracellular recordings reported here is small, but these provide important confirmation of earlier findings of the enhancement of synaptic currents and of putative calcium currents in neurons in hippocampal slices during low pi o treatment (Huang et al. 1997). In the previous study (Huang et al. 1997), cells were patch-clamped in the whole-cell configuration. During such whole-cell recordings, much of the cytoplasm is replaced by the solution that fills the pipette. We now used high-resistance sharp electrodes so that the interior of the cell was much less disturbed. In addition, the membrane potential was not clamped, allowing the cell to generate its physiological voltage signals. Similarly to the synaptic currents recorded with the patch-clamp method in the previous study (Huang et al. 1997), the EPSPs recorded with sharp electrodes increased in amplitude when pi o was lowered. In addition, in some trials, slow waves made an appearance that could be triggered by EPSPs (Fig. 7) or by depolarizing pulses (Fig. 8). These slow waves were probably generated by the slow currents that were evident in the patch clamp recordings made under similar hypoosmotic conditions (Huang et al. 1997). They are quite similar to published recordings of dendritic calcium-dependent action potentials (e.g., Llinas and Sugimori 1980a; Wong et al. 1979). Slow waves could perhaps also be generated by persistent sodium current, INa,P (Crill 1996; Llinas and Sugimori 1980b). This is, however, unlikely because in isolated neurons INa,P is depressed in low pi o and in low [NaCl]o, as may be expected from the reduction of the driving potential caused by lowering [Na+]o (Somjen 1999b).

Previously we reported that brief, sudden exposure of isolated neurons to very strongly hypoosmotic solution results in a suppression of voltage dependent K+ and Ca2+ currents (Somjen et al. 1993). This finding seems contrary to the apparent enhancement of putative calcium currents seen in neurons in hippocampal slices reported here (Figs. 7 and 8) and in Huang et al. (1997). The seeming contradiction may result from different rate, duration, and strength of the hypoosmotic exposure. The hypotonicity experienced by cells in the slice was milder, had a much more gradual onset, and lasted much longer than that imposed on dispersed cells in a previous study (Somjen et al. 1993). In recent trials, it appears that mild-to-moderate, gradual reduction of pi o causes depression of potassium currents but enhances voltage-dependent calcium currents in isolated hippocampal neurons (Somjen 1999b). In intact neurons, in situ depression of K-currents could disinhibit dendritic calcium currents. The initial transient decrease of both baseline [Ca2+]i and stimulus-evoked Delta [Ca2+]i responses (Fig. 6) could represent a mild form of the "channel shutdown" reported previously (Somjen et al. 1993). During prolonged lowering of pi o, the mechanisms raising [Ca2+]i and enhancing ICa appear to overtake the shutdown of calcium channels.


    ACKNOWLEDGMENTS

This research was supported by the Netherlands Organization for the Advancement of Pure Research (NWO) and by National Institute of Neurological Disorders and Stroke Grant NS-18670.


    FOOTNOTES

Address for reprint requests: G. G. Somjen, Dept. of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710.

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 10 June 1999; accepted in final form 1 October 1999.


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