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 |
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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 (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
o by ~70 mOsm caused
"resting" [Ca2+]i as well as synaptically
or directly stimulated transient increases of calcium activity
(
[Ca2+]i) to transiently decrease and then
to increase. In dissociated cells, lowering
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
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
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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INTRODUCTION |
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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 (
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
o (Chebabo et al. 1995a
;
Huang et al. 1997
; Huang and Somjen 1995
;
Rosen and Andrew 1990
). Low
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
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 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
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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METHODS |
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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 o was lowered by deleting NaCl, and in
isoosmotic low NaCl medium, mannitol replaced NaCl. Sharp electrodes
(impedance >80 M
) 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
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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RESULTS |
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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 o (at equimolar [NaCl]o), whereas in other cells two different
levels of
o were compared. Exposures lasted
5-7 min. The order of presentation of the test solutions was varied
among cells.
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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 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
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 (
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
(
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
o
solution of the same [NaCl]o.
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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
o (
[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
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 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,
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
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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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
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
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
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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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 o (NaCl
reduced by 40 mM;
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
[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 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
[Ca2+]o transients were
plotted against the number of action potentials fired, the average
[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
o it increased in all 5 neurons measured,
from a mean control value of 108 ± 26 to 140 ± 31 M
.
Next it returned to 109 M
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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DISCUSSION |
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Lowering 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 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
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
o.
In an earlier study, we found that interstitial calcium concentration
in hippocampal slices decreased when hippocampal slices were exposed
either to low 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
o. When external calcium was removed, lowering
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
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
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
[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
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
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
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
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
[Ca2+]i responses (Fig. 6) could represent
a mild form of the "channel shutdown" reported previously
(Somjen et al. 1993
). During prolonged lowering of
o, the mechanisms raising
[Ca2+]i and enhancing ICa appear to overtake
the shutdown of calcium channels.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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