Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Müller, Michael and
George G. Somjen.
Intrinsic Optical Signals in Rat Hippocampal Slices During
Hypoxia-Induced Spreading Depression-Like Depolarization.
J. Neurophysiol. 82: 1818-1831, 1999.
In
interfaced rat hippocampal slices spreading depression (SD) and
hypoxia-induced SD-like depolarization are associated with increased
light reflectance and decreased light transmittance, indicating
increased light scattering. By contrast, mild hypotonicity or
electrical stimulation decrease light scattering, which is usually
taken to be caused by cell swelling. This difference has been
attributed to experimental conditions, but in our laboratory moderate
osmotic challenge and SD produced opposite intrinsic optical signals
(IOSs) in the same slice under identical conditions. To decide whether
the SD-induced IOS is related to cell swelling, we investigated the
effects of Cl transport inhibitors and Cl
withdrawal on both light reflectance and transmittance, as well as on
changes in interstitial volume and tissue electrical resistance. In
normal [Cl
]o, early during hypoxia, there
was a slight decrease in light reflectance paired with increase in
transmittance. At the onset of hypoxic SD, coincident with the onset of
cell swelling (restriction of TMA+ space), the IOS signals
suddenly inverted, indicating sharply increased scattering. The
SD-related IOSs started in a single spot and spread out over the entire
CA1 region without invading CA3. Application of 2 mM furosemide
decreased IOS intensity. When [Cl
]o was
substituted by methylsulfate or gluconate, the SD-related reflectance
increase and transmittance decrease were suppressed and replaced by
opposite signals, indicating scattering decrease. Yet Cl
withdrawal did not prevent cell swelling measured as shrinkage of
TMA+ space. The SD-related increase of tissue electrical
resistance was reduced when bath Cl
was replaced by
methylsulfate and almost eliminated when replaced by gluconate. The
TMA+ signal is judged to be a more reliable indicator of
interstitial space than tissue resistance. Neither application of
cyclosporin A nor raising [Mg2+]o depressed
the SD-related reflectance increase, suggesting that Cl
flux through mitochondrial "megachannels" may not be a major factor
in its generation. Fluoroacetate poisoning of glial cells (5 mM)
accelerated SD onset and enhanced the SD-induced reflectance increase
threefold. This suggests, first, that glial cells normally moderate the
SD process and, second, that neurons are the predominant generators of
the light-scattering increase. We conclude that light scattering by
cerebral tissue can be changed by at least two different physical
processes. Cell swelling decreases light scattering, whereas a second
process increases scattering. During hypoxic SD the scattering increase
masks the swelling-induced scattering decrease, but the latter is
revealed when Cl
is removed. The scattering increase is
Cl
dependent, nevertheless it is apparently not related
to cell volume changes. Its underlying mechanism is as yet not clear; possible factors are discussed.
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INTRODUCTION |
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Intrinsic optical signals (IOS) of brain tissue
slices have been found useful in visualizing neuronal excitation
without using fluorescent dyes. They are closely associated with
neuronal stimulation, excitotoxicity, epileptiform activity, cell
swelling, as well as spreading depression (SD) and hypoxic SD-like
depolarization, and, depending on the experimental conditions, they
show marked differences with respect to their intensity, time course,
and direction (Aitken et al. 1998; Basarsky et
al. 1998
; Holthoff and Witte 1996
;
MacVicar and Hochman 1991
; Meierkord et al.
1997
). IOSs have usually been attributed to changes in light
scattering by the tissue, and they have been taken to be a measure of
average cell volume (e.g., Lipton 1973
; Ørskov
1935
; reviewed by Aitken et al. 1999
). Besides
cell volume changes, there appear, however, to be additional mechanisms
that also may contribute to the generation of IOSs (Kreisman et
al. 1995
; Meierkord et al. 1997
). The present study therefore focuses on the mechanism of IOSs associated with hypoxic SD and investigates their correlation to cell swelling.
The scattering of light influences the translucence of an object and
the amount of light reflected from its surface in opposite sense:
increased scattering decreases light transmittance but increases light
reflectance, whereas decreased scattering causes reciprocally opposite
changes. By contrast, altered absorption of light affects transmittance
and reflectance in the same direction, for if light energy is converted
either into heat or during a photochemical process, both reflected and
transmitted light will decrease. Also, changes in the geometry of an
interfaced tissue slice would alter reflected and transmitted IOS in
the same sense. Reducing the radius of curvature of the surface causes
the slice to resemble a convex lens, changing the angles of both
refraction and reflection so as to divert light away from the detector
looking down on the slice, no matter whether the light came through the tissue, or was incident on it (Aitken et al. 1999;
Born and Wolf 1970
; Kreisman et al.
1995
).
Previous reports show that mild to moderate hypotonicity reduces the
intensity of light reflected from the surface of a tissue slice while
increasing the light transmitted through the tissue (Andrew and
MacVicar 1994; Lipton 1973
). This is in line
with the expected reduction in light scattering due to hypotonic cell swelling. Electrical stimulation of cerebral tissue also causes reduced
scattering in the excited region, and the resulting IOSs are diminished
by application of furosemide or Cl
withdrawal,
confirming that they are related to cell swelling (Andrew and
MacVicar 1994
; Holthoff and Witte 1996
;
Lipton 1973
; MacVicar and Hochman 1991
).
In contrast, during SD, a process known to be associated with marked
cell swelling (Hansen and Olsen 1980
; Jing et al.
1994
), light scattering increases, and this change is much
greater than the scattering decrease seen under milder challenges
(Aitken et al. 1998
; Martins-Ferreira and
Oliveira Castro 1966
; Müller and Somjen
1998c
; Snow et al. 1983
).
In an attempt to resolve this seeming paradox, Kreisman et
al. (1995) reported that light transmittance changes induced by osmolarity invert sign when the bath level is lowered so that the
previously submerged slice comes to lie at the interface between liquid
and gas. Kreisman et al. (1995)
concluded that in
submerged slices changes in scattering within the tissue generate the
IOS, whereas in slices lying at a gas-liquid interface the refraction at the slice surface determines the signal. Hypotonic swelling causes
the slice to bulge, i.e., it shortens the radius of curvature, resulting in less light being collected by the detector. When the slice
is submerged, this surface-optical effect is abolished because the
refractive indices of the bath and the tissue are similar.
Kreisman et al. (1995)
also pointed out that the
SD-related increase of scattering has been observed in interfaced
slices, whereas the swelling-related decrease was reported for
submerged tissues. In keeping with these arguments, Basarsky et
al. (1998)
observed increased light transmittance (decreased
scattering) during SD in submerged hippocampal slices. It should be
remembered, however, that the isolated retina preparation produces a
marked increase of light scattering during SD, which is easily seen
with the naked eye, even though it is completely submerged in bathing fluid (Martins-Ferreira and Oliveira Castro 1966
).
The strikingly large and sudden optical changes during hypoxic SD
in hippocampal slices begin at the same time as the sudden intra- and
extracellular potential shifts signaling SD onset. In an earlier study
we monitored light reflectance of hippocampal slices under a variety of
manipulations designed to influence SD-related biophysical changes. We
found that pharmacological inhibition of hypoxic SD by blockade of the
major Na+ and Ca2+ pathways
also prevents the hypoxia-induced optical changes (Müller and Somjen 1998c). The simultaneous onset of the electrical and optical signs as well as the cell swelling during normoxic and hypoxic
SD (Jing et al. 1994
) seemed to reinforce the earlier assumption that the IOS reflects cell swelling, even if the scattering increase was opposite to the scattering decrease associated with moderate hypotonicity (Snow et al. 1983
; Turner
et al. 1995
).
In the experiments presented here, we examined the relationship of the
optical signals to Cl fluxes and to cell volume
changes detected by the indicator-dilution technique (Hansen and
Olsen 1980
; Phillips and Nicholson 1979
) as well
as tissue resistance measurements (Freygang and Landau 1955
). Contrary to expectation, we found that the hypoxic
SD-induced light-scattering increase is not related to cell swelling or
the resulting restriction in extracellular space, even though it is dependent on Cl
flux. Metabolic poisoning of
glial cells accelerated the onset of hypoxic SD and intensified the
associated optical signals, indicating that viable glial cells are not
required for the generation of the optical signal. Positive evidence
for a major contribution of mitochondrial swelling to the generation of
the optical signal was not obtained.
Parts of this study have been published in abstract form
(Müller and Somjen 1998a,b
, 1999
;
Somjen and Müller 1999
).
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METHODS |
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Preparation
Hippocampal tissue slices were prepared from male Sprague-Dawley rats (95-210 g body wt; 4-6 wk old). The rats were decapitated under ether anesthesia, and the brain was rapidly removed from the skull and placed in chilled artificial cerebrospinal fluid (ACSF) for 1-2 min. One hippocampus was isolated, and transverse slices of 400-µm thickness were cut using a tissue chopper. Slices were transferred to an interface recording chamber and were left undisturbed for 90 min. The recording chamber was kept at a temperature of 34.5-35.5°C. It was continuously aerated with 95% O2-5% CO2 (400 ml/min) and perfused with oxygenated ACSF (1.5 ml/min). Hypoxia was induced by switching the chamber's gas supply to 95% N2-5% CO2. To protect the slices from drying out and to prevent oxygenation from the air during hypoxic episodes, the slice chamber was covered by a lid with a small (2 cm2) opening for the positioning of the electrodes. Exchange of the bathing solution and diffusion of applied drugs into the slice took ~15 min.
Solutions
The ACSF had the following composition (in mM): 130 NaCl, 3.5 KCl, 1.25 NaH2PO4, 24 NaHCO3, 1.2 CaCl2, 1.2 MgSO4, and 10 dextrose; aerated with 95%
O2-5% CO2 to adjust pH to
7.4. When TMA+-sensitive microelectrodes were
used to monitor changes in extracellular space, 1.5 TMA+ (tetramethylammonium-chloride, ICN) was
added. In low Cl solution either methylsulfate
or gluconate replaced all but 3.9 mM Cl
(as
CaCl2 and TMA-Cl). Furosemide (Sigma),
4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS; TCI), and
fluoroacetate (Sigma) were directly dissolved in ACSF.
4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; Sigma) was
dissolved in distilled water before being added to ACSF.
Glibenclamide (RBI) was dissolved in dimethyl sulfoxide (DMSO;
Sigma) to prepare a 100-mM stock solution (stored at
20°C), and
diazoxide (RBI) was dissolved in 0.1 M NaOH (20 mM stock solution;
20°C). Cyclosporin A (Calbiochem) was dissolved in absolute ethanol (25 mM stock solution;
20°C). Immediately before the experiment, 0.1 ml DMSO were added to 0.1 ml cyclosporin A stock solution, and this
mixture was then added to well-oxygenated ACSF. Final ethanol and DMSO
concentrations were 0.02% for cyclosporin A solutions and 0.1% DMSO
for glibenclamide solutions.
Microelectrodes
Single-barreled glass microelectrodes for extracellular
recordings were pulled from thin-walled borosilicate glass (TW150F-4, WPI) using a horizontal puller (Flaming Brown, P-80/PC). They were
filled with ACSF, and their tips were broken to a final resistance of
5-10 M. Changes in extracellular space were quantified by measuring
changes in the background concentration of the membrane-impermeable TMA+ ion (indicator-dilution technique) (see also
Hansen and Olsen 1980
; Nicholson and Phillips
1981
; Phillips and Nicholson 1979
). TMA+-sensitive microelectrodes were made from
double-barreled theta type capillaries (GCT 200-10, Clark
Electromedical Instruments). The ion-sensitive barrel was silanized by
60-min exposure to hexamethyldisilazane vapors (HMDS 98%, Fluka;
vaporized at 40°C). Silanization of the reference barrel was
prevented by perfusion with compressed air (1.5 bar). The tip of the
ion-sensitive barrel was filled with the Corning 477317 K+ ion exchanger (IE190, WPI) and backfilled with
150 mM TMA-Cl + 10 mM N-(2-hydroxyethyl)
piperazine-N'-(2-ethanesulfonic acid) (HEPES; Sigma), pH
7.4. The reference barrel contained 150 mM NaCl + 10 mM HEPES, pH 7.4. Mean electrode resistances of the reference and ion-sensitive barrel
were 20-40 M
and 80-110 M
, respectively.
TMA+-sensitive electrodes were calibrated before and after each experiment by detecting the response generated in standard TMA+ solutions (0, 0.1, 0.5, 1, 5, 10, 50, and 100 mM). The ionic strength of the calibration solutions was held constant and corresponded to extracellular conditions. The electrodes did not respond to replacing O2 by N2, and because the backfill solution of the ion-selective barrel did not contain K+, [K+]o changes had little effect on electrode potential. In the presence of 1 mM TMA+, increasing [K+]o from 3.5 to 50 mM induced an electrode response of only 0.3 ± 1.3 mV. Average slope of the electrodes was 60.6 ± 2.7 mV/decade TMA+, and their detection limits were 0.13 ± 0.10 mM TMA+ (mean ± standard deviation, n = 23).
Electrical recordings
Electrode signals were referred to an Ag/AgCl reference
electrode embedded in 2% agar in 3 M KCl. They were recorded by a DC
amplifier (constructed locally) and digitized by a TL-1/Lab Master
acquisition system at sampling rates of 10 Hz using the Axotape V2
software (Axon Instruments). Because electrodes were calibrated to
TMA+ concentrations and the
TMA+ activity coefficient was held constant,
changes in [TMA+]o could
directly be calculated from the electrode responses using the
electrodes' averaged slope of pre- and postexperiment calibration. Changes in relative interstitial volume (ISV) during hypoxia were calculated as a percent of "control" according to the formula (Dietzel et al. 1980)
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Changes in electrical tissue resistance during hypoxia were measured
using the "four-electrode" method (Freygang and Landau 1955; Li et al. 1968
; Traynelis and
Dingledine 1989
). Two monopolar microwire stimulation
electrodes, representing anode and cathode, were used to deliver
constant current pulses of 3-5 µA amplitude and 20-30 ms duration
every 10 s; the same pulse was used throughout any one experiment.
Stimuli were generated by photoelectric stimulus isolation units (Grass
PSIU6). The resulting voltage deflections were measured differentially
by a pair of extracellular glass microelectrodes (filled with low
[Cl
] ACSF) positioned between the two
stimulation electrodes. All four electrodes were placed in a straight
line in stratum (st.) radiatum; distance of the two stimulation
electrodes was at ~1 mm, and that of the recording electrodes was at
~500 µm (Fig. 6A). The amplitudes of the voltage
deflections were determined at the steady-state level, and changes in
electrical tissue resistance were normalized to the prehypoxic tissue
resistance of the respective slice in normal solution. Because the
electrode distances varied somewhat in the different slices, only these
relative changes are reported.
Recording of the intrinsic optical signal
Light reflectance or transmittance was recorded in st. radiatum of the CA1 region using a 12-bit charge-coupled device (CCD) camera (Princeton Instruments, PentaMAX System). Slices were illuminated by white light at angles of 20° relative to the line of vision of the microscope objective (reflectance), or 180° (transmittance). Size of the digitized pictures was 248 × 190 pixel; their optical resolution was 45 µm2/pixel. Images were recorded at 3-s intervals and 0.2-s exposure time and transferred to an IBM-compatible computer. Hypoxia-induced changes were visualized by digital image subtraction and were referred to the image taken before SD onset. The optical changes were displayed in a 256 gray-scale mode covering a full-scale range of ±15% brightness changes and quantified for a representative rectangular region of interest close to the microelectrode recording DC potential and/or TMA+ signal (Fig. 2B). The initial time course of the IOS was found to be linear until the maximum brightness was approached (1st 30-40 s). We therefore fitted the relative changes during the first 30 s (1st 10 images) after onset of the reflectance increase by linear regression and used the resulting slope as a measure for the invasion speed of the CA1 region. Photoshop 4.0 software (Adobe) was used for graphic processing.
Statistics
The data were obtained from 98 rats. Because most experiments lasted between 4 and 8 h, usually only one slice could be used from each brain. Numerical values are represented as means ± standard deviation. Significance was tested using a two-tailed, unpaired Student's t-test, and a significance level of 5%. In the diagrams, significant changes are marked by asterisks (*P < 0.05; **P < 0.01). Statistical calculations and linear regressions were done with the Excel 7.0 or QuattroPro 3.0 software.
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RESULTS |
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Electrical signs of SD in different regions of hippocampal slices
The hippocampal CA1 region is highly vulnerable to oxygen
withdrawal and has a low threshold for SD initiation. Simultaneous recording of DC potential changes during severe hypoxia from different hippocampal areas showed that hypoxic SD can occur in all hippocampal regions, although it occasionally spared CA3 (Fig.
1). It was always most prominent in CA1
and the adjacent subiculum; a spread from CA1/CA2 into CA3, or vice
versa, was never observed. Mean amplitudes for the rapid negative DC
potential deflection (Vo) were as
follows: CA1,
14.5 ± 7.3 mV; CA2/3,
6.3 ± 5.4 mV; CA3,
6.5 ± 7.5 mV; CA4,
6.9 ± 6.1 mV; dentate gyrus,
7.8 ± 6.0 mV; subiculum,
15.3 ± 7.9 mV; (mean ± standard deviation; each n = 19).
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Optical changes
Mapping the hypoxia-induced increase in light reflectance to the
various slice regions of a different set of 37 control slices showed
that severe hypoxia induced optical changes in the CA1 region of every
slice investigated. In 17 of 37 slices, the IOS spread from the CA1
area into the subiculum, but only in one slice did it enter the dentate
region. Optical changes in CA2 occurred in 11 of the investigated
slices, but a further spread from CA1 into CA3 could never be observed
(see also Aitken et al. 1998, 1999
;
Müller and Somjen 1998c
; Obeidat and Andrew
1998
). In some slices, however, an IOS appeared in CA3
independently from that seen in CA1. Because the electrical and optical
signs of hypoxic SD usually occurred first and were most intense in CA1
region, in many of the trials we limited the optically recorded field to the CA1 region to improve spatial resolution.
In the CA1 region of interfaced rat hippocampal tissue slices, severe
hypoxia induced biphasic changes in light reflectance at the slice
surface. In all slices, hypoxic SD was preceded by a slowly progressing
reflectance decrease, which continued until SD onset. This slow
decrease was replaced by a sudden, large increase in light reflectance,
which coincided exactly with the hypoxic Vo, beginning 1.5-3 min after
oxygen withdrawal (Fig. 2A)
(see also Aitken et al. 1998
; Müller and
Somjen 1998c
). The reflectance increase started in a small
focus in stratum (st.) radiatum and spread along the entire CA1 region.
It was most pronounced in st. radiatum, followed by st. oriens, whereas
st. pyramidale was mostly spared (Fig. 2B) (Aitken et
al. 1998
; Müller and Somjen 1998c
). Under
control conditions the reflectance increase in st. radiatum averaged
8.1 ± 4.2% (n = 70), occurred with an initial rate of rise of 0.21 ± 0.13%/s and reached its maximum intensity and spread within 45 ± 16 s after onset of the reflectance
increase (Figs. 2B and 3).
Recovery of the optical changes was much slower than that of the
Vo and was incomplete 3 min after
reoxygenation.
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When transmitted instead of reflected light was recorded, the direction
of the optical changes was the reverse of those seen in reflectance.
Thus during hypoxia, but before SD onset, there was a slow and moderate
increase in transmittance, whereas during hypoxic SD light
transmittance decreased by 11.2 ± 5.8% (n = 5). The time courses of transmittance and reflectance changes were identical (Fig. 3) (see also Aitken et al. 1999). The
opposite changes in reflected and transmitted light indicate that
hypoxic SD indeed increases light scattering rather than modifying
light absorbance or changing the geometry of the reflecting/refracting surface.
Control experiments confirmed that, similarly to SD-induced
Vo, repeating hypoxic episodes at
45-min intervals did not alter the intrinsic optical signals. In a
given slice, the reflectance increase started in the same focus during
four consecutive hypoxic episodes, and significant changes in intensity
and spreading speed could not be detected (n = 8, Fig.
3A).
Cl dependence of the hypoxia-induced scattering
increase
As marked cell swelling coincides with normoxic and hypoxic SD
(Hansen and Olsen 1980; Jing et al.
1994
), one might suspect that these cell volume changes are the
mechanism responsible for the generation of the optical changes during
SD, as has indeed been suggested in previous reports (Aitken et
al. 1998
; Basarsky et al. 1998
;
Müller and Somjen 1998c
; Snow et al.
1983
). Because Cl
fluxes via either
Cl
channels or secondary active
Cl
transport systems play a key role in volume
regulation and cell swelling (for review see Kaplan et al.
1996
; Strange 1993
), we investigated whether the
established Cl
transport inhibitors furosemide,
DIDS, and DNDS as well as Cl
substitution by
methylsulfate modulate the intrinsic optical signals associated with
hypoxic SD.
The inhibitors and the Cl substitute were
applied for 45 min before hypoxic SD was induced. In well-oxygenated
slices their administration hardly affected the light reflectance
baseline. Although in untreated control slices the tissue reflectance
decreased during 45 min by 0.64 ± 1.01% (n = 8),
Cl
transport inhibition caused the following
reflectance changes: furosemide,
0.28 ± 1.55%
(n = 7); DIDS, 1.74 ± 1.3% (n = 6); DNDS, 0.32 ± 1.49% (n = 8);
Cl
substitution by methylsulfate, 0.19 ± 2.02% (n = 8).
During SD and hypoxic SD the extracellular Cl
concentration drops by at least 60 mM (Nicholson 1984
;
Nicholson and Kraig 1981
), due to
Cl
influx into neurons and glial cells
(Jiang et al. 1992
). Accordingly, we observed
distinctive changes in both electrical and optical signs of hypoxic SD,
when hypoxia was induced in the presence of the
Cl
transport inhibitors or the
Cl
substitute. We have previously found that
furosemide and DIDS depress the amplitude of the hypoxic
Vo by 19 and 33%, respectively (Müller and Somjen 1998a
,b
). Similarly to its
effect on
Vo, furosemide also
depressed the hypoxia-induced intrinsic optical signal. The intensity
of the reflectance increase was reduced by 28.6 ± 14.0%, and it
evolved 34.3 ± 14.1% slower (n = 8, Fig. 3B). DIDS and DNDS (0.1 mM) did not affect hypoxia-induced
intrinsic optical signals (n = 7 and n = 5, respectively).
The most remarkable effect occurred when 98% of extracellular
Cl was replaced by the membrane-impermeable
methylsulfate or gluconate anions. In methylsulfate-substituted
solution the amplitude of the hypoxic
Vo decreased by only 32.8 ± 11.2% (n = 11), but the SD-associated reflectance
increase was completely suppressed. Instead, hypoxic SD was now
paralleled by a smaller but still substantial light reflectance
decrease, which was 76.3 ± 17.5% less intense than the
reflectance changes seen under control conditions (n = 6; Figs. 3C and 4).
Correspondingly, the decrease in light transmittance during SD was also
reversed in methylsulfate-based low Cl
solutions, and it was replaced by brightening of the slices in transmitted light during SD, being 76.2 ± 55.1% less intense
than the previously recorded transmittance decrease in ACSF
(n = 5, Fig. 3D). Both the
Vo depression and the inversion of
the optical signs of SD by Cl
withdrawal were
completely reversible; 90 min after readdition of
Cl
hypoxic SD increased light reflectance by
126.5 ± 29.8% of control (n = 6), and light
transmittance decreased by 263.1 ± 126.6% of control
(n = 5; Figs. 3, C and D, 4).
Substitution of Cl
by gluconate also
consistently and reversibly abolished the reflectance increase during
SD and replaced it by a reflectance decrease (n = 5).
|
TMA+ space changes compared with the optical signals in
normal solution and after Cl withdrawal
Because the Cl dependence of the optical
changes suggested that cell swelling could be responsible for the
generation of the IOS, we simultaneously recorded interstitial
TMA+ concentration
([TMA+]o),
Vo, and light reflectance. Because
a constant extracellular Cl
concentration was
an absolute requirement (especially in low Cl
solutions), we were not able to apply TMA+ by
either pressure of iontophoretic ejection (Dietzel et al. 1980
; Nicholson 1992
; Nicholson and
Phillips 1981
) and were methodologically restricted to the
analysis of TMA+ background changes. These
measurements were first performed in normal
[Cl
]o solution and then
repeated on the same slice 45 min after substitution of extracellular
Cl
by methylsulfate, and again 45 min after
restoring [Cl
]o. As
shown in Fig. 2B, the reflectance changes were analyzed in a
region close to the recording electrode.
During the initial phase of hypoxia, before onset of the SD-like
Vo, a slow negative DC potential
shift, a slight decrease in interstitial volume (
9.5 ± 3.6%),
and a decrease in light reflectance (decreased scattering) occurred
simultaneously. In normal
[Cl
]o, hypoxic SD began
1.4 ± 0.4 min after oxygen withdrawal. It was indicated by the
sudden
Vo of
18.2 ± 5.3 mV,
and it coincided with a 63%
[TMA+]o increase
(corresponding to a reduction of interstitial space by 37.2 ± 9.5%) and an 9.8 ± 4.1% (n = 13) increase in
light reflectance. Both the optical changes and the cell swelling
evolved more slowly than the
Vo.
Although the
Vo reached its peak
within 2.8 ± 3.3 s, cell swelling and reflectance increase
reached their maximum intensity 35.4 ± 22.2 s and 49 ± 17 s (n = 13) after SD onset. Measured at the
half-amplitude level, optical changes and
[TMA+]o increase lasted
107 ± 44 s and 81.8 ± 26.8 s, respectively, compared with 33.7 ± 10.9 s (n = 13) for the
Vo. During the posthypoxic recovery
phase, [TMA+]o usually
undershot the baseline by 13.7 ± 6.9% (Fig.
5A), which probably results
from posthypoxic cell shrinkage. It could also indicate loss of
TMA+ from tissue spaces during the preceding
period of [TMA+]o
elevation, by diffusion of TMA+ into unaffected
tissue areas and the bath.
|
To estimate exchange between tissue and bath, two types of experiments were conducted. In one series, hypoxia was maintained for 15 min after SD onset. During such prolonged hypoxia, [TMA+]o gradually declined by 38.0 ± 11.5% (n = 5). Assuming that the swollen state of cells was maintained until reoxygenation was started, this slow decline represents loss of TMA+ from the tissue to the bath. As soon as oxygen was readmitted, the decline of [TMA+]o toward baseline was markedly accelerated. In a second set of trials, bath [TMA+] was raised and then lowered while tissue [TMA+] was recorded in the presence of normal oxygen. TMA equilibration in well-oxygenated slices was faster than in hypoxic slices. The slower diffusion in the hypoxic tissue is explained by the restricted diffusion path due to cell swelling. In slices previously incubated for 45 min in a bath containing 3 mM TMA+, the half-decay time for reequilibration in 1.5 mM TMA+ medium averaged 204 ± 40.3 s (n = 5). By contrast, topical local application of 3 mM TMA+ ACSF resulted in shorter half-decay times (99.2 ± 52.4, n = 6), indicating, as expected, that equilibration with the bath is much slower when [TMA+]o is raised diffusely in wide areas or the entire slice, as was the case in the trials involving hypoxic SD.
Because withdrawal of extracellular Cl caused
the reversal of the optical signs of SD (Figs. 3 and 4), we now asked
whether it also prevented cell swelling. In the presence of normal
oxygen, substitution of extracellular Cl
by
methylsulfate transiently widened TMA+ space by
9.0 ± 5.0% (n = 8), following which
[TMA+]o returned to its
baseline. This slow apparent recovery was probably in large part due to
diffusion of TMA+ from the bath into the tissue,
with a possible contribution of volume regulation especially in glial
cells. On restoration of [Cl
]o, the calculated
extracellular space decreased from its perhaps still widened volume by
19.1 ± 18.5% (n = 6).
Contrary to expectation, when hypoxic SD was induced 45 min after
Cl had been replaced by methylsulfate, the
hypoxia-induced decrease in calculated TMA+ space
was not significantly affected, averaging 101.9 ± 26.6% of
control (n = 8; Figs. 5B and
6C). However, an undershot of the [TMA+]o baseline
during the recovery phase did not occur. In these slices, as before,
the SD-related reflectance increase was suppressed and replaced by
reflectance decrease, whereas the
Vo amplitude decreased by 31.2 ± 9.5%. When hypoxic SD was induced 45 min after restoring
[Cl
]o the
Vo amplitude averaged
12.7 ± 4.3 mV, a reflectance increase of 8.7 ± 3.5% was again
observed and extracellular space decreased by 88.8 ± 24.4% of
control (n = 8).
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The unchanged amount of cell swelling in low Cl
solutions might indicate that the cell membranes became permeable for
methylsulfate during hypoxia. We therefore repeated these experiments
using the even less permeable Cl
substitute,
gluconate. Administration of gluconate-based low Cl
solution reversibly widened
TMA+ space by 11.0 ± 1.8%
(n = 5). In the presence of gluconate, the hypoxic
Vo amplitude decreased by 33.3 ± 18.4%, and SD occurred 33.5 ± 24.7% (n = 5)
earlier than under control conditions. In the same five slices the
SD-associated reflectance increase inverted into a 79.2 ± 13.8%
less intense reflectance decrease, similar to the effect of
methylsulfate. Again, the increase in
[TMA+]o was not
suppressed, averaging 103.0 ± 5.6% of control and indicating unchanged cell swelling (Fig. 6C). An undershot of the
[TMA+]o baseline during
the recovery phase was not observed.
Changes in electrical tissue resistance during hypoxic SD
To confirm the unexpected observation that cell swelling still
occurs following nearly complete Cl
substitution, we measured the electrical tissue resistance during hypoxic SD in normal and low Cl
solutions (Fig.
6). Restriction of extracellular space during SD is well known to
increase the tissue resistance (Hoffman et al. 1973
;
Marshall 1959
; Van Harreveld and Ochs
1957
), even though the input resistance of neurons, and to a
lesser degree that of glial cells, decreases. In our experiments,
hypoxic SD in control solution was associated with an increase in
tissue resistance by 35.5 ± 10.1% (n = 11). The
time course of the resistance changes resembled the time course of the
[TMA+]o changes, and
following reoxygenation the tissue resistance also undershot the
prehypoxic baseline level (Fig. 6B). In normal oxygen,
Cl
substitution by methylsulfate and
restoration of normal
[Cl
]o did not
significantly affect the electrical tissue resistance, which averaged
108.2 ± 17.6% and 93.9 ± 24.3% of control
(n = 6), respectively. When hypoxic SD was induced in
methylsulfate-containing solution, the tissue resistance still clearly
increased, the increase averaging 57.7 ± 11.0% of control
(n = 6), which confirms that Cl
substitution does not abolish cell swelling during severe hypoxia. After restoration of
[Cl
]o, the increase in
tissue resistance during SD averaged 113.0 ± 34.7% of control
(n = 6).
Cl substitution by gluconate had, however, a
different effect on tissue resistance. On administration of gluconate,
the tissue resistance at first became almost double and then decreased
again somewhat, averaging after 45-min treatment 126.5 ± 5.4% of
control. During hypoxic SD the tissue resistance increase was
abolished, averaging only 2.4 ± 1.6% of control
(n = 5). Yet the increase in
[TMA+]o, measured in the
same slices with the TMA+-sensitive electrode
positioned in between the two extracellular recording electrodes, was
not diminished (108.4 ± 5.8% of control; n = 5).
Restoring normal [Cl
]o
decreased the tissue resistance to 99.2 ± 7.0% of its control level and during hypoxia tissue resistance increased again, by 93.0 ± 28.3% (n = 5) of control, whereas
[TMA+] increased by 84.4 ± 11.4% of control.
Contribution of glial cells
Severe hypoxia caused the most prominent scattering changes in st.
oriens and st. radiatum, i.e., in layers rich in glial processes. To
decide whether glial cells contribute to the generation of the optical
signals, we impaired glial function by metabolic poisoning with
fluoroacetate. Fluoroacetate is selectively taken up by glial cells and
metabolized into fluorocitrate, which arrests the glial tricarboxylic
acid cycle (citric acid cycle) and thus blocks glial metabolism
(Clarke and Nicklas 1970; Hassel et al. 1992
). Over the course of 3-4 h, hippocampal glial cells
gradually depolarize,
[K+]o slightly increases,
and pHo decreases. Neurons are, however, at first
almost completely unaffected and severe neuronal damage expressed as
loss of electrogenic activity does not occur before 8 h of
fluorocitrate treatment (Largo et al. 1996
,
1997a
). In our experiments application of 5 mM
fluoroacetate for up to 6 h shortened the time to onset of hypoxic
Vo. The reduction in time to SD
onset averaged 20.6 ± 8.3% after 1.5 h and 28.9 ± 21.6% after 6 h fluoroacetate treatment, whereas
Vo amplitude and duration were not
significantly affected (n = 9; Figs.
7 and
8A). The intensity of the
SD-associated scattering increase was greatly enhanced, reaching after
6 h fluoroacetate treatment 296.6 ± 197.1% of its control
amplitude (n = 6; Figs. 7 and 8B).
|
|
Failure to demonstrate a role of mitochondrial swelling in IOS generation
Hypoxia is known to induce the mitochondrial permeability
transition and result in mitochondrial swelling (Lemasters et
al. 1997). The trigger event of mitochondrial swelling appears
to be the activation of the so-called "megachannel" in the outer mitochondrial membrane, which enables anion and cation fluxes into the
mitochondrion (Colombini 1994
; Lemasters et al.
1997
). Interestingly, mitochondrial swelling is highly
Cl
dependent (Azzone et al.
1976a
,b
), and it has been reported to cause scattering changes
in hippocampal slices (Johnson et al. 1998
). We
therefore attempted to test whether known blockers of the mitochondrial
permeability transition can prevent the hypoxia-induced scattering
increase. Application of 5 µM cyclosporin A (60 min) an inhibitor of
the mitochondrial "megachannel" (Colombini 1994
; Lemasters et al. 1997
; Qian et al. 1997
),
increased the
Vo amplitude by
25.0 ± 20.8% and shortened its duration by 21.2 ± 16.5%.
It failed, however, to depress the SD associated reflectance increase (n = 5). Raising extracellular
Mg2+ to 5 mM (Kristal and Dubinsky
1997
) reversibly delayed the onset of the hypoxic
Vo by 46.0 ± 33.3% and
decreased focal excitatory postsynaptic potential (fEPSP) amplitudes by
58.5 ± 26.5%, but again the SD associated scattering increase
was not reduced (n = 7).
No evidence for the involvement of ATP-sensitive K+ channels in SD or its IOS
The involvement of ATP-sensitive K+ channels
in the hypoxic hyperpolarization of CA1 pyramidal neurons preceding
hypoxic SD is controversial (Erdemli et al. 1998;
Fujimura et al. 1997
). Modulation of these channels by
45-min applications of the specific inhibitor glibenclamide (0.1 mM;
n = 7) or the activator diazoxide (0.1 mM;
n = 6) did not affect the hypoxic
Vo or the associated optical
signals. Therefore these channels are unlikely to play a crucial role
in the triggering of hypoxic SD or the generation of the IOS.
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DISCUSSION |
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Dual nature of hypoxia-induced IOSs
Intrinsic optical signals associated with severe hypoxia are characterized by biphasic optical changes. An initial decrease in reflected light changes at the onset of the SD-like event to a much more pronounced reflectance increase. Accordingly, when transmitted light was recorded, an initial increase in transmittance was followed by a transmittance decrease (Fig. 3). Because, during both phases, reflectance and transmittance were consistently influenced in opposite sense, these optical signals may be attributed to changes in light scattering within the tissue.
Kreisman et al. 1995 described an inversion of the sign
of transmitted light change that depended on the bath level, the signal changing as the slice was either submerged in solution or exposed at
the upper surface to the gas phase. In our experiments the level of the
bath was not changed, and the slices were constantly located at the
liquid/gas interface; therefore these changes in experimental
conditions cannot explain the reversal of the optical changes that
coincides with SD onset. It could be argued, however, that during the
first few minutes of hypoxia cell swelling was mild and the dominant
IOS was reduced scattering within the tissue, whereas during SD the
severe swelling caused the surface of the slice to bulge, and the
"lensing" effect described by Kreisman et al. (1995)
became dominant. This is also unlikely, because a reduced radius of
curvature would have diminished the intensity of both, reflected and
transmitted light (see INTRODUCTION) but, in fact,
reflected light consistently increased during SD in normal solution, in
agreement with earlier reports (Aitken et al. 1998
; Martins-Ferreira and Oliveira Castro 1966
;
Müller and Somjen 1998c
; Snow et al.
1983
). We therefore conclude that during the initial, pre-SD
phase of hypoxia light scattering decreases, whereas SD itself
coincides with a marked increase in scattering within the tissue.
Dissociation of electrical signals, IOS and cell swelling following
Cl withdrawal
Although the sudden hypoxic
Vo, the scattering increase, and
the restriction of extracellular space coincided in control solutions,
these measures were differently affected when hypoxia was induced
following Cl
substitution. The extracellular
potential shift was partially depressed, and the scattering increase
was completely suppressed, but cell swelling was unaffected.
The persistence of the hypoxic
Vo in low
Cl
solutions and the fact that SD-related
depolarization of pyramidal neurons is not reduced by
Cl
withdrawal (Müller and Somjen
1998b
) indicate that Cl
fluxes are not
responsible for the massive potential shifts or their regenerative
nature. The observed 31% decrease in
Vo amplitude (Figs. 4 and 5) is
similar to that reported by Do Carmo and Martins-Ferreira (1984)
for normoxic SD in isolated retina. It could be due to the moderate widening of extracellular space following
Cl
withdrawal (as indicated by the decrease in
[TMA+]o), which may
dampen extracellular potential shifts, or desynchronized neuronal
depolarization as was reported for epileptiform discharges in the
presence of furosemide (Hochman et al. 1995
).
The most significant observation was, however, the separation of
SD-related scattering increase and cell swelling. Contrary to
expectation, after Cl withdrawal, SD-related
cell swelling, measured as the
[TMA+]o increase, was
unchanged. Even though it is an indirect measure, the indicator
dilution method is a well-established technique (Dietzel et al.
1980
; Hansen and Olsen 1980
; Nicholson
1992
; Nicholson and Phillips 1981
;
Phillips and Nicholson 1979
). Of the different possible
marker ions (TEA, TMA,
-naphtalenesulfonate, choline), TMA+ has been deemed to be the best
(Nicholson 1992
), even though it was occasionally
reported that TMA+ may sometimes enter neurons
and glial cells (Ballanyi et al. 1990
; Jing et
al. 1994
; Nicholson 1992
), thereby causing an
underestimation of volume changes.
Unlike the TMA+ signal, the well-known
SD-related increase in tissue resistance (Marshall 1959;
Van Harreveld and Ochs 1957
) was reduced when
methylsulfate replaced Cl
, and even more when
the substitute was gluconate. The question is, which measurement,
TMA+ or resistance, is the more reliable. For the
following reasons we believe that TMA+ space
gives a better indication. Tissue resistance can be an accurate
indicator of cell volume only as long as the applied current flows in
interstitial space and not through cells. Okada et al.
(1994)
have shown that, in the turtle cerebellum, a
considerable fraction of the test current in fact does flow through the
transcellular path. Although the proportion undoubtedly varies among
tissues, this source of error cannot be neglected. The error becomes
worse, if the interstitial resistance increases while transcellular
resistance does not. We found that the resistivity of
methylsulfate-based ACSF is ~30% higher than that of normal
solution, and for gluconate-based ACSF the resistivity increase was
73%. Moreover, with normal oxygen supplied, both methylsulfate and
gluconate substitution of Cl
caused the
prehypoxic interstitial volume to increase by 9-10%. This shifts the
resistance ratios in the "voltage divider" and as a result an equal
(absolute) increase of cell volume would restrict the fractional
interstitial volume to a (relatively) lesser degree. It should be noted
that exposure to hyperosmotic solutions also results in a much greater
increase in TMA+ space than decrease of tissue
resistance, and it is the TMA+ signal that
correlates with the depression of synaptic transmission (Huang
and Somjen 1995
).
The degree of cell swelling during hypoxic SD measured here (37% ISV
restriction in st. radiatum) was less pronounced than that reported by
Jing and coworkers (1994; their value: 66% ISV restriction in st. pyramidale). This discrepancy may be due to the
different cytoarchitectonic layers investigated or technical differences. Because we based estimates on baseline
[TMA+]o instead of
repeated iontophoretic ejection of the indicator, diffusion of
TMA+ during its elevated level into tissue areas
not affected by SD and into the bath might have resulted in an
underestimation of cell swelling.
Possible mechanisms of cell swelling in low Cl
solution
If we accept that the TMA+ signal accurately
indicates the fractional interstitial volume, it appears that the
prominent cell swelling during hypoxic SD was not prevented by
Cl withdrawal. However, the absence of an
undershot of the [TMA+]o
baseline following SD recovery in low Cl
solutions might indicate impairment of regulatory volume decrease in
the presence of methylsulfate and gluconate.
The unchanged amount of cell swelling is surprising because there can
be little doubt that, in the presence of normal
[Cl]o, hypoxic cell
swelling depends on Cl
uptake into cells
(Strange 1993
;Van Harreveld 1966
). We
must ask what drives the influx of water in the absence of chloride. It
appears unlikely that the remaining
[Cl
]o (3.9 mM) is
sufficient to account for the unchanged amount of cell swelling.
Because most Cl
channels are also permeable to
bicarbonate, the observed cell swelling in low
Cl
solutions could be due to bicarbonate influx
into neurons and glial cells, which of course would disturb
intracellular pH. Additional but less likely factors may be enhanced
membrane permeability to larger anions during SD, permitting entry of
methylsulfate and gluconate. Phillips and Nicholson
(1979)
determined the penetration of several anions during
normoxic SD in cerebellum, but methylsulfate and gluconate were not
among those tested. Products of anaerobic metabolism could raise the
osmolarity of the cytosol, but this effect is probably too slow to
explain the explosive SD-like swelling. Even less probably,
interstitial space could have shrunk without cell swelling, in the
manner of a squeezed sponge, but no force is known that would be able
to perform the squeezing.
Unlike SD-related cell swelling, stimulation-induced volume changes in
neocortical slices are abolished when Cl is
substituted by gluconate (Holthoff and Witte 1996
).
Holthoff and Witte (1996)
attributed the swelling mainly
to uptake of KCl, probably by glial cells. Because fluoroacetate and
fluorocitrate do not diminish electrophysiological signs of normoxic or
hypoxic SD (Largo et al. 1997a
,b
) (and see
RESULTS in this paper), it seems likely that SD-related
swelling resides mainly in the dendritic arbor of neurons. It seems
therefore that high K+-induced glial swelling is
Cl
dependent, whereas SD-related neuronal
swelling is not. Apparently at variance with our observations,
Lipton (1973)
reported that hypoxia-induced reflectance
changes were abolished when Cl
was substituted
by glucuronate. In the absence of electrophysiological recording it is, however, doubtful that his McIlwain-style submerged slices underwent SD-like depolarization. Gradual cell swelling and
depolarization can occur without the SD-like event, and the slow
swelling observed by Lipton (1973)
may be
Cl
dependent, whereas the abrupt SD-related swelling is not.
At least two mechanisms induce light-scattering changes
Intrinsic optical signals caused by neuronal excitation are about
an order of magnitude less intense than the optical changes associated
with SD. The most remarkable difference is, however, that they show
opposite direction. Although neuronal excitation decreases light
scattering, SD is paralleled by a scattering increase. The
Cl dependence of the decrease of light
scattering and its sensitivity to furosemide have already been reported
(Hochman et al. 1995
; Holthoff and Witte
1996
; Lipton 1973
; MacVicar and Hochman
1991
) and were taken to support the association of these
signals with cell swelling. The results reported here show that this is
not the case for the scattering increase associated with hypoxic SD. Cl
withdrawal caused the separation of the
electrical and optical signs of SD. The reflectance increase was
completely suppressed, whereas cell swelling was not affected (Fig. 5).
Furthermore, st. radiatum and st. pyramidale show a comparable amount
of cell swelling during SD (Jing et al. 1994
;
Pérez-Pinzón et al. 1995
), but increased
scattering occurs only in the dendritic layers. We therefore conclude
that cell swelling and increased light scattering are not correlated.
The evidence is compelling, however, that the reduced scattering seen
in mild to moderate hypotonia and in response to electrical stimulation
of the tissue is indeed related to increased cell volume (Aitken
et al. 1999; Andrew and MacVicar 1994
;
Holthoff and Witte 1996
; Lipton
1973
; MacVicar and Hochman 1991
).
Kreisman et al. (1995)
reported, however, that, unlike
in submerged slices, in slices at a liquid-gas interface light
transmittance decreased with reduced osmolarity. This was not the case
in our interface chamber where withdrawal of 40 mM NaCl decreased light
reflectance by 2.9 ± 2.1% (n = 10, data not
shown) indicating decreased scattering. In a different study hypotonic
exposure increased transmittance and decreased reflectance of
hippocampal slices in two interface chambers with differing optical
systems, as long as osmolarity was reduced by not more than 75 mosm/kg
(Aitken et al. 1999
; D. Fayuk, unpublished
observations). It therefore seems very likely that the mild decrease of
light scattering seen during hypoxia before SD, as well as the much
more marked scattering decrease observed during SD in low
Cl
solutions (Figs. 3 and 4), are caused by cell
swelling. Surprisingly, we also found (Aitken et al.
1999
) that the decreased light scattering typical of mild to
moderate hypotonia inverted into an increase when bath osmolarity was
reduced to almost half normal (
144 mosm/kg). Recurrent waves of SD
induced by the very low osmolarity were accompanied by waves of
scattering increase on an already elevated baseline.
We conclude that there are at least two processes that influence light
scattering in opposite sense. The dilution of macromolecules in the
cytosol during cell swelling is probably responsible for the reduction
of light scattering consistently observed in cell suspensions and
tissue slices exposed to mild to moderate hypotonicity, as well as in
neural tissue stimulated electrically. A second process of unknown
origin causes an increase in light scattering, and it appears to
dominate when hippocampal slices are exposed to severe hypotonicity or
undergo SD-like depolarization. Removal of Cl abolishes
the second process and unmasks the first, revealing that the
swelling-induced scattering decrease is latently present even during
hypoxic SD.
Possible generators of the light-scattering increase
Glial poisoning by fluoroacetate (Clarke and Nicklas
1970; Hassel et al. 1992
) did not prevent the
initiation and the spread of hypoxic SD but rather enhanced the
tissue's susceptibility to SD, as has already been reported for
normoxic SD (Largo et al. 1997a
,b
). The progressive
impairment of glial function gradually increased the intensity and
extension of the light-scattering increase during SD, until it reached
threefold intensity after 6 h (Figs. 7 and 8). We therefore
conclude that generation of the SD-associated scattering increase does
not require intact glial cells or a functioning glial "syncytium."
The huge increase of the SD-related optical signal after glial
poisoning suggests that intact functioning glia dampens the process
that causes the scattering increase. In any event, neurons and not
glial cells appear to be mostly responsible for the scattering increase.
The predominance of SD-related optical changes in st. radiatum and st.
oriens, as well as their persistence during glial poisoning do suggest
that the underlying mechanisms occur mainly in the dendritic neuronal
processes, or the interstitial space in between. Even though the
refraction by cell membranes embedded between cytosol and interstitium
may be minor, when many fine processes are closely packed, their
scattering effect may be multiplied. A measure of geometric complexity
of interstitial space is the tortuosity (Nicholson and Phillips
1981). Pérez-Pinzón et al. (1995)
have determined tortuosity as well as interstitial volume fraction
during oxygen and glucose withdrawal in hippocampal slices. Tortuosity
in the pyramidal layer of CA1 region did not significantly change
during SD-like depolarization, and this suggests that it did not
contribute to the optical signal. Because, however, st. radiatum was
not explored by Pérez-Pinzón et al. (1995)
,
an effect by tortuosity cannot be ruled out. In fact increased
tortuosity may even be suggested by the hindered
TMA+ diffusion observed during maintained hypoxia.
The scattering increase is not due to the bulging or "lensing" of
the swollen slice surface reported by Kreisman and coworkers (1995), first, because cell swelling was unaltered after
Cl
withdrawal while the scattering increase was
reversibly abolished and, second, because bulging of the surface should
alter reflectance and transmittance in the same sense (see
INTRODUCTION). Besides, the strong increase of
reflectance during SD in submerged retinae (Martins-Ferreira and
Oliveira Castro 1966
) confirms that scattering can increase in
neural tissue independently from surface optics. In the submerged
slices maintained at 33-34°C by Basarsky et al. (1998)
, the scattering increase was, apparently, not activated during SD. Polischuk and colleagues (1998)
and
Obeidat and Andrew (1998)
report that light
transmittance decreases in such slices when oxygen and glucose are
withdrawn at 37.5°C, and they relate this change from increased to
decreased transmittance (i.e., from decreased to increased scattering)
to irreversible damage, expressed as beading of neuronal dendrites.
Because in our experiments the SD-related scattering increase evolved
and reversed rapidly without permanently impairing synaptic or other
neuronal function, dendritic injury cannot be its cause.
The most obvious two sources of light scattering in live tissue are
macromolecules in cytosol, and cell organelles. Influx of water into
cells dilutes macromolecules and is the likely reason for the decreased
scattering during swelling. Swelling of organelles, on the other hand,
would increase the surfaces of scattering particles, and it could be
the mechanism of Cl-dependent scattering increase. As a
possibility we investigated the involvement of mitochondrial swelling,
which is triggered by hypoxia (Lemasters et al. 1997
)
and may induce scattering changes in hippocampal slices (Johnson
et al. 1998
). Application of cyclosporin A and elevated
[Mg2+]o effectively reduce or even prevent
mitochondrial swelling in isolated mitochondria or permeabilized cells
(Kristal and Dubinsky 1997
; Qian et al.
1997
). In our experiments, however, these agents failed to
reduce the SD-associated optical signals. Even though the shortened SD
duration (
21.2 ± 16.5%) may suggest a protective effect of
cyclosporin A, its penetration into the tissue slice and uptake into
cells are, however, questionable and leave the outcome inconclusive.
Besides, it is not clear whether mitochondrial swelling is already
triggered immediately after SD onset or whether it is the consequence
of continued hypoxia only.
Besides mitochondrial swelling and beading of dendrites, hypoxia and
ischemia are known to induce a variety of ultrastructural changes, such
as chromatin clumping and nucleolar condensation, cytoskeletal and
microtubule breakdown resulting in cell shape changes, membrane
blebbing and increased membrane fluidity as well as disruption of
endoplasmic reticulum and Golgi apparatus (Allen et al.
1989; Kwei et al. 1993
; Tanaka et al.
1999
). Each of these changes could modify light scattering, but
it should be considered that severe structural changes are usually
irreversible, while the scattering increase associated with SD reversed
within a few minutes.
The physical mechanism of the scattering increase remains, for now,
unclear. Although the scattering increase does not occur without the
sudden hypoxia-induced potential shifts (Müller and Somjen
1998c), it now became apparent that the
Vo of hypoxic SD can proceed without an
increase in light scattering. Our most important finding is that, even
though the light-scattering increase that coincides with hypoxic SD is
Cl
dependent, it is not generated by cell swelling, or by
the resulting restriction of extracellular space.
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ACKNOWLEDGMENTS |
---|
We thank Dr. D. A. Turner for providing his CCD camera.
This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-18670.
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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 19 January 1999; accepted in final form 22 June 1999.
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REFERENCES |
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