Department of Physiology and Neuroscience, New York University Medical Center, New York, New York 10016
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ABSTRACT |
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Tao, Lian
Effects of osmotic stress on dextran diffusion in rat neocortex studied
with integrative optical imaging. This study investigated how
dextran (Mr = 3,000) diffused in rat cortical
slices when the osmolarity of the bathing artificial cerebrospinal
fluid was altered by varying the NaCl content. The apparent diffusion
coefficient, D*, was measured in the neocortex region using
fluorescent molecules and the integrative optical imaging (IOI) method.
The main results were: 1) the value of D* in rat
neocortex in the isotonic (300 mOsm) artificial cerebrospinal fluid at
34°C was D* = 0.68 ± 0.01 × 106
cm2 s
1 (mean ± SE, n = 78) and it could be changed within minutes by varying the extracellular
osmolarity. 2) Hypotonic stresses up to
100 mOsm decreased
D* by 35% and were fully reversible when the slices were
returned to the isotonic medium. Further hypotonic stress to
150 mOsm
caused further decrease in D* but after removal of the
stress, D* overshot its control value. 3)
Hypertonic stress of +50 mOsm increased D*, but the maximum
reversible increase in D* was only 15%. Further hypertonic
stress (to +200 mOsm) did not cause any further increase in
D* and, after removal of the stress, D* undershot
the control value. The changes in D* are thought to be
related to volume changes of cells in tissue: hypotonic solutions
caused cell swelling, resulting in reduced extracellular space and
compressed extracellular matrix so that the dextran diffusion was more
hindered. Hypertonic solutions had the opposite effect. Recordings of
extracellular field potentials in the hippocampal CA1 region
demonstrated that, on return to the isotonic solution after exposure to
an extreme hypotonic or hypertonic stress, the neurons retained their
ability to generate synaptic responses.
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INTRODUCTION |
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The diffusion of molecules through the
extracellular space (ECS) of the brain plays a critical role in
fundamental biological processes such as the movement of metabolic
substrates (Fellows et al. 1992) and volume
transmission. Volume transmission also is called extrasynaptic or
nonsynaptic transmission, in contrast to classical synaptic
transmission, and has been proposed to be another mechanism of
intercellular communication via substance diffusion in the ECS.
(Agnati et al. 1995
; Bach-y-Rita 1994
;
Fuxe and Agnati 1991
; Syková 1997
).
The diffusion of molecules in ECS also plays an important role in
clinical applications such as the delivery of drugs (Krewson et
al. 1995
) and diffusion-weighted magnetic resonance imaging
(Latour et al. 1994
; Szafer et al. 1995
).
One of the most important parameters that characterize the diffusion
properties of molecules in ECS is the apparent diffusion coefficient
(ADC), denoted by D*. The ADC is defined by the diffusion equation
C*/
t = D*
2C*, where C* is the
local average concentration of the diffusing molecules in brain tissue
(Nicholson and Phillips 1981
; Nicholson and
Syková 1998
). It is known that for small ions, such as
tetramethylammonium (TMA+, Mr = 74),
D* is relatively constant across a spectrum of brain tissues
(Nicholson 1993
; Nicholson and Syková
1998
; Syková 1997
). It also is known that
in the same brain region, different macromolecules, such as dextrans
(Nicholson and Tao 1993
), albumins (Tao and
Nicholson 1996
), and N-(2-hydroxypropyl)
methacrylamide polymer (HPMA) (Vargová et al.
1998
), may have different values of D*, even when
their molecular weights are similar. This indicates that the ADC of a
molecule depends not only on its molecular weight but also on other
properties, including shape, radius of gyration, electric charge, etc.
These molecular attributes, together with the physico-chemical properties of the ECS, determine the interactions between the molecule
and the structure of the ECS. Consequently, if the diffusing molecule
is kept the same and the ECS properties are varied, we may anticipate
that the ECS constraints encountered by the diffusing molecule will
change, thereby resulting in changes in the ADC. This raises the
following questions. For a given molecule, especially a macromolecule,
in a given tissue, can the ADC be changed experimentally? If yes, how
quickly or how effectively can such changes be made? What are the
limits of the changes? The answers to these questions are not only
helpful to understanding the characteristics of the interactions
between the molecule and the ECS structure but also useful in many
clinical applications.
To explore these issues, the ADC of fluorescent dextran molecules (Mr = 3,000) was measured in rat cortical slices using the integrative optical imaging (IOI) method. The slices were subjected to different osmotic stresses induced by changing the NaCl content of the bathing medium.
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METHODS |
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Brain slices were obtained from Sprague Dawley rats of either
sex (~150 g). The rats were anesthetized deeply with pentobarbital sodium (65 mg/kg) and decapitated, and the cerebrum was removed. Coronal slices with a thickness of 400 µm were cut at the interaural 5- to 6-mm planes (Paxinos and Watson 1986). After
incubation in artificial cerebrospinal fluid (ACSF, defined later) at
room temperature, the slice was transferred to the measurement chamber kept at 34 ± 1°C. The chamber was perfused with ACSF and the
slice was submerged 3 mm below the fluid surface. The flow of ACSF was maintained at 1 ml/ min using a peristaltic pump. Diffusion
measurements were made in cortical layers III, V, and VI, but no
differences were noted in different layers. A total of 26 rats were
used with between two and four slices from each rat. Several
measurement sequences were made on each slice, and the value of
n cited in statistics represents the number of measurement sequences.
The IOI system as well as the related theory and data analysis
techniques have been detailed elsewhere (Nicholson and Tao 1993; Tao and Nicholson 1995
), so here is only a
brief description. The measurement chamber was mounted on the stage of
a compound epi-fluorescent microscope. Dextran
(Mr = 3,000), tagged with the fluorescent dye
Texas Red, (D-3329, Molecular Probes, OR) was dissolved in 154 mM NaCl
saline to form a 1-mM solution. The fluorescent molecules were ejected
into the brain slice with a brief pressure-pulse from a micropipette,
which was inserted 200 µm below the slice surface. The diffusing
molecules were imaged with a ×10 water-immersion objective. Before
each dextran ejection, an image was taken and saved as the background
image. After each ejection, a sequence of 10 images was recorded at an
interval of 10 s using a cooled CCD camera and transferred
directly to a personal computer, where the background image was
subtracted from the diffusion images. Then intensity profile was taken
through the ejection point in each image and fitted with the
theoretical expression for a diffusion image using the nonlinear
Levenberg-Marquardt algorithm to extract the apparent diffusion
coefficient D* (Nicholson and Tao 1993
).
The normal isotonic ACSF had the following composition (in mM): 115 NaCl, 5 KCl, 35 NaHCO3, 1.25 NaH2PO4, 1.3 MgCl2, 1.5 CaCl2, and 10 D-glucose, resulting in an
osmolarity of 300 mOsm. The osmolarities of all the ACSFs were
determined with a freezing point depression osmometer (Osmette,
Precision Systems, MA), and the errors in the osmolarities were within
±5 mOsm. The ACSFs were gassed continuously with 95%
O2-5% CO2 to maintain a pH of 7.5. Three
hypotonic ACSF solutions were used representing decreases of 50,
100, and
150 mOsm from the isotonic ACSF. They were obtained by
reducing the NaCl content of the isotonic solution. Three hypertonic
ACSF were used representing increases of +50, +100, and +200 mOsm from
the isotonic ACSF, and they were obtained by increasing NaCl content.
The measurement chamber initially was perfused with the isotonic ACSF,
then a brain slice was transferred into the chamber and allowed to
equilibrate with the bath for ~10 min. Two or three sequences of
diffusion images were recorded to obtain the control value of
D*. To study the effect of an osmotic stress, the isotonic ACSF was replaced with either a hypotonic or a hypertonic solution. Test experiments showed that the ADC began to change in ~5 min after
a change of solution and reached a steady value within 10 min.
Therefore the recording of diffusion images under the osmotic stress
was started after 10 min and several sequences of images were taken.
Then the isotonic ACSF was switched back, and 10 min later more image
sequences were recorded to study the recovery behavior. As an example,
Fig. 1 shows the diffusion images of the
fluorescent dextran in a cortical slice recorded with the IOI method.
In the figure, each of the three rows shows an image sequence taken
after an ejection. The three sequences were taken from the same slice
but in different solutions. The middle row was the
image sequence in the normal isotonic solution (300 mOsm). The
top row was the sequence in a mild hypotonic solution
(50 mOsm), whereas the bottom row was the sequence in a
mild hypertonic solution (+50 mOsm).
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The viability of the slices was assessed by recording extracellular
field potentials from slices in the same chamber, under superfusion
conditions identical to those for the imaging experiments. The
recordings were performed in the hippocampal CA1 region instead of
neocortical region because the pathway formation is well defined in the
hippocampus and allows a reliable recording of monosynaptic transmission and monitoring the tissue excitability. The field potentials were evoked by stimulating the Schaffer collaterals with
brief current pulses (25 mA, 30 µs) from a bipolar twisted wire
electrode and recorded from the pyramidal layer with a glass micropipette filled with 2 M NaCl. As an example, Fig.
2A shows field potentials
recorded for a hypotonic stress (100 mOsm), whereas B
shows the potentials for a hypertonic stress (+200 mOsm). The top
row in the figure shows the initial control recordings, which have
a single population spike typical for a healthy unchallenged slice
(Andrew and Macvicar 1994
; Ballyk et al.
1991
; Chebabo et al. 1995a
; Saly and
Andrew 1993
). When the hypotonic stress was applied, multiple
population spikes appeared with the amplitude several times greater
than the initial control signal (Fig. 2A, middle). This
phenomenon has been reported previously (Andrew and Macvicar
1994
; Ballyk et al. 1991
; Chebabo et al.
1995a
; Saly and Andrew 1993
). Direct
intracellular recordings from the CA1 and CA3 neurons in rat
hippocampal slices have shown that the evoked excitatory postsynaptic
potential (EPSP) was not affected by hypotonic stress and, therefore,
the greater amplitude of population spike has been attributed to
enhanced synchronization of neuron discharge due to reduction of ECS.
(Ballyk et al. 1991
; Saly and Andrew
1993
). As argued later in the present paper, the size of ECS
was reduced markedly under an extreme hypotonic stress. After removal
of the hypotonic stress, the single population spike was restored but
with a smaller amplitude (Fig. 2A, bottom), indicating that
the neurons had become less excitable. In contrast, the hypertonic stress eliminated the population spike completely (Fig. 2B,
middle); however, after removal of the stress, multiple population
spikes appeared with greater amplitudes (Fig. 2B, bottom),
indicating the neurons had become hyperexcitable. Each trace in the
figure is an average of four field recordings taken 30 min after a
change of solution, which was comparable with the length of time for the image experiments. From the field potential recording, it was
evident that after exposure to an extreme hypotonic or hypertonic stress, the neurons did not come back their initial condition completely. However, synaptic transmission clearly was restored.
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RESULTS |
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Normal isotonic condition
The apparent diffusion coefficient D* of the dextran in
the neocortex under the normal isotonic condition served as the control value for the experiment. Measurements were made in each cortical slice
under isotonic ACSF flow. These data are shown on each quantile plot in
Figs. 3 and
4. A quantile plot shows the complete
distribution of a set of data. Each point in the plot represents a
measured D*. The ordinate of the point is just the value of
the D* while the abscissa represents the fraction of data
that have values lower than this value (Chambers et al.
1983). The mean value of the ADC for all the experiments was
D* = 0.68 ± 0.01 × 10
6
cm2 s
1 (n = 78) at 34°C. The values
for D* here and elsewhere in RESULTS are quoted
as means ± SE.
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Hypotonic conditions
To study the effect of hypotonic stress, the isotonic ACSF was replaced with a hypotonic solution (Fig. 3). The value of D* began to decrease after ~5 min and reached a steady value in ~10 min. For some slices, the measurement of D* in the hypotonic solution was repeated during a period of 90 min, but no further changes were observed after the first 10 min. Consequently, the data presented here were acquired during the period of 10-30 min after initiating the flow of hypotonic ACSF.
At the conclusion of the hypotonic stress, the isotonic solution was restored (Fig. 3). The value of D* began to recover in ~5 min and reached a steady value in ~10 min. Again, prolonged measurements showed that no significant changes occurred after the first 10 min after the restitution of the normal solution, and so the recovery data presented here were acquired during the first 10-30 min after the isotonic solution was restored.
Figure 3A shows the quantile plot of the data acquired under
the mild hypotonic stress of 50 mOsm. The mean value of D*
was 0.53 ± 0.02 × 10
6 cm2
s
1 (n = 23), representing a 22% decrease
from the normal D* value. Figure 3A also shows
the data acquired after the slices had recovered from the hypotonic
stress; under these conditions, D* = 0.69 ± 0.02 × 10
6 cm2 s
1
(n = 17), which was very close to the normal value,
showing that the changes in D* caused by this hypotonic
stress were reversible.
When the hypotonic stress was increased, by reducing osmolarity by
100 mOsm, D* decreased further (Fig. 3B). Under
these conditions, D* = 0.44 ± 0.02 × 10
6 cm2 s
1 (n = 34), representing a 35% decrease from the control D* value. After recovery from the hypotonic stress (Fig. 3B),
D* = 0.67 ± 0.02 × 10
6
cm2 s
1 (n = 28), which was
still close to the control value, indicating that the changes in
D* caused by the stronger hypotonic stress were still reversible.
As more hypotonic stress was applied, D* decreased further.
Figure 3C shows the data acquired under the most extreme
hypotonic stress that was used, 150 mOsm. In this case,
D* = 0.38 ± 0.02 × 10
6
cm2 s
1 (n = 18), which
represented a 45% decrease from control. However, after such a strong
hypotonic stress was removed, D* no longer returned to the
initial value (Fig. 3C) but instead exhibited an overshoot
so that a value of D* = 0.78 ± 0.03 × 10
6 cm2 s
1 (n = 17) was measured. For some slices, D* repeatedly was
measured for 2 h, but the overshoot was unchanged.
Hypertonic conditions
The time courses of the changes under hypertonic conditions were similar to those under the hypotonic ones so the data were acquired during the period of 10-30 min after initiating the hypertonic ACSF flow.
Figure 4A shows the quantile plot of the data acquired under
the weak hypertonic stress of +50 mOsm. The mean value of the ADC was
D* = 0.78 ± 0.02 × 106
cm2 s
1 (n = 19), representing
a 15% increase over control. After recovery from the hypertonic
stress, D* = 0.69 ± 0.03 × 10
6
cm2 s
1 (n = 13), which was
very close to the initial value, indicating that the change in
D* was reversible.
As more hypertonic stress was applied, D* did not increase
further. Figure 4, B and C, shows the data
acquired under hypertonic stresses of +100 and +200 mOsm, respectively.
The values of D* were 0.77 ± 0.03 × 106 cm2 s
1 (n = 9) and 0.78 ± 0.02 × 10
6 cm2
s
1 (n = 27), respectively. They both
represented about a 15% increase in D* compared with
control, indicating that the increase in D* caused by
hypertonic stress has an upper limit, at least over the range tested
here. However, after the stronger hypertonic stresses were removed,
D* no longer returned to the control value. Figure 4,
B and C, shows the data acquired after the
isotonic ACSF flow was restored, revealing an undershoot in
D* in each case. The values of the undershoots were
D* = 0.62 ± 0.02 × 10
6
cm2 s
1 (n = 13) and
D* = 0.58 ± 0.02 × 10
6
cm2 s
1 (n = 21),
respectively. These values were significantly lower than the control
value. For some slices, D* was repeatedly measured for
2 h, but the undershoot was sustained.
The data obtained under hypotonic stresses (Fig. 3) and under hypertonic stresses (Fig. 4) are summarized in Fig. 5. Note in the figure that while the ADC could decrease dramatically with osmolarity, it could only increase a very limited amount from its control value no matter how much hypertonic stress was applied. Also note that the maximum reversible change under hypotonic stress was also much greater than that under hypertonic stress.
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The goal of this study was to investigate how a moderately large
molecule diffused in brain tissue when the ECS osmolarity was changed.
The main results were: 1) the ADC, D*, of dextran (Mr = 3,000) in brain tissue could be changed
within minutes by varying the extracellular osmolarity. 2)
Hypotonic stress, brought about by reduced NaCl content, decreased
D*. Decreases of 35% in D* (100 mOsm) were
fully reversible when the tissue was returned to control medium.
Further hypotonic stress caused further decrease in D*, but
after removal of the stress, D* overshot its control value.
3) Hypertonic stress, induced by excess NaCl content,
increased D*. The maximum reversible increase in
D* was 15%, (+50 mOsm). Further hypertonic stress did not
cause any further increase in D*, and after removal of the
stress D* undershot the control value.
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DISCUSSION |
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To understand the experimental results, I suggest the following
explanations. Many studies have demonstrated that varying the
extracellular osmolarity can alter the volume of cells in tissue. One
recent study (Chebabo et al. 1995b), using ion-selective microelectrodes to measure extracellular concentration change of a
probe ion TMA+ or tetraethylammonium (TEA+),
estimated that when hippocampal tissue slices were exposed to a severe
hypotonic stress (
180 mOsm), the extracellular volume was reduced to
25% of its control value, corresponding to a mean cell volume increase
of
11%. Another recent study (Krizaj et al. 1996
),
using ion-selective microelectrodes to measure TMA+
diffusion in the isolated turtle cerebellum combined with wet- and
dry-weight measurements of tissue, showed that the volume fraction of
the ECS (defined as the ratio of the volume of the ECS to volume of
whole tissue in a small representative region) changes when
extracellular osmolarity is varied by altering the NaCl content of the
ACSF. That study further demonstrated that such changes in ECS volume
fraction primarily are brought about by water moving between the extra-
and intracellular compartments. Tosteson and Hoffman (Tosteson
1964
; Tosteson and Hoffman 1960
) proposed a
theoretical model for regulation of cell volume when external NaCl is
varied. The model was based on the interaction of electrical and
osmotic effects. According to this model, when the rate of the Na-K
pump is fixed, the cell volume is inversely proportional to the total
extracellular ion concentration. Moreover, when the extracellular ion
ratio [Na+]o/[K+]o
is reduced, cell volume increases (Hoppensteadt and Peskin 1992
). Therefore it is established, experimentally and
theoretically, that a hypotonic stress achieved by reduced
[NaCl]o causes cell swelling, whereas a hypertonic stress
achieved by excess [NaCl]o causes cell shrinking.
It is known that, under the normal osmotic condition, the ECS occupies
~20% of the total volume of brain tissue (Nicholson 1993; Nicholson and Phillips 1981
;
Nicholson and Syková 1998
). The ECS harbors a
matrix consisting of long-chain glycoproteins and glycosaminoglycans,
including hyaluronate (Bignami et al. 1993
;
Margolis and Margolis 1993
; Ruoslahti
1996
), and there is renewed interest in a particular form of
the matrix called the perineuronal net (Celio and Blumcke
1994
). The matrix may be sufficiently dense to form an
obstructive polymer, which would restrict the diffusion of molecules
(Ogston and Sherman 1961
). In the present experiments,
the ECS is the space within which the dextran diffuses because the cell
membranes are impermeable to the dextran over the periods of typical
measurements. When cells swell, the ECS will become narrower and
therefore may hinder the passage of molecules. More importantly, the
swelling cells could compress the matrix, making it denser and more
obstructive to diffusing molecules, resulting in a lower ADC. This is
true especially for larger molecules the dimensions of which may
approach the average path width within the matrix. The converse of
these arguments would apply in hypertonic medium, as the cells shrank.
Therefore the change in the ADC of macromolecules in some way reflects
the volume change of cells in tissue. Obviously the quantitative
relation between the changes in ADC and cell volume is complicated, and
it may depend heavily on the type of tissue and the type of
macromolecule (Nicholson et al. 1998
).
The asymmetric behavior of the ADC under hypotonic and hypertonic
conditions is worth noting. Although the ADC could decrease dramatically with osmolarity, it can only increase a very limited amount. Based on the argument that the change in ADC reflects the
change in cell volume, this result may lead to the hypothesis that the
decrease in cell volume caused by hypertonic stress is very limited.
This interesting and important point has not been predicted by Tosteson
and Hoffman's theoretical model. Therefore an extension to their model
is needed. The extension might involve another regulation mechanism,
which is activated when the cell volume decreases below a certain
critical level. The regulation mechanism would prevent any further
decrease in cell volume so that cell functions could be maintained. A
possible candidate for the regulation mechanism is the uptake channels
through which KCl can be taken up rapidly (Cserr et al.
1987a,b
; Gullans and Verbalis 1993
; Law
1994
). As KCl accumulates, the intracellular osmolarity
increases and the water efflux is stopped, preventing further decreases
in cell volume. Besides the possible regulation mechanism involving ion
redistribution across the membrane, physical constraints imposed by the
cytoskeleton and the extracellular matrix also may limit the shrinkage
at a certain point.
It has been known that cultured brain cells respond to hypotonic stress
by initial swelling, which is followed by the regulatory volume
decrease (RVD) within minutes. Similarly, their initial shrinkage
responding to hypertonic stress is followed, within minutes, by the
regulatory volume increase (RVI) (Ballanyi and Grafe
1988; Hoffmann 1987
). However, the question of
whether RVD or RVI also occurs in brain slices is still not entirely
resolved. Although one recent study showed some data supporting RVI in
rat hippocampal slices (Chebabo et al. 1995b
), another
more detailed study provided evidence against the regulatory volume
changes in rat hippocampal slices (Andrew et al. 1997
).
Because of the nature of the IOI technique, it is not possible to
obtain a continuous time course of the change in ADC. However, as
mentioned before, for some slices the ADC measurement was repeated
during a period of 90 min. It has been found that all the decreases and
increases of ADC occurred within the first 10 min after a stress had
been applied and the changes were monotonic. If the decreases and
increases of ADC indeed reflects the changes in cell volume, the
monotonic decreases and increases of ADC means the RVD or RVI has not
been seen in this study.
Although the present experiment only demonstrated that the ADC is a
function of ECS osmolarity, from the above explanation, it is very
likely that ADC also depends on several physiological conditions that
affect cell volume. This explanation leads to the possibility that
measuring the ADC of macromolecules could be a useful method to study
the changes in volume of cells in tissue. This is pertinent to clinical
issues that arise when the ECS is reduced in size, during ischemia,
anoxia, or edema for example (Syková 1997). From
the preceding explanation, it is also apparent that under hypotonic
conditions, large molecules, especially those that have the dimensions
comparable to the average path width of the extracellular matrix, are
going to be more hindered than small ones. This may have consequences
for therapeutic intervention, not only in stroke but also, for example,
in the delivery of nerve growth factor and similar molecules to treat
Alzheimer's disease (Krewson et al. 1995
).
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ACKNOWLEDGMENTS |
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I am grateful to Dr. C. Nicholson for very helpful discussion and Dr. S. Hrabetova for assistance with the field potential recording.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-28642 to C. Nicholson.
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FOOTNOTES |
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Address for reprint requests: Dept. of Physiology and Neuroscience, New York University Medical Center, 550 First Ave., New York, NY 10016.
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 13 August 1998; accepted in final form 20 January 1999.
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REFERENCES |
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