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The Journal of Neuroscience, September 15, 1998, 18(18):7189-7199
Imaging Spreading Depression and Associated Intracellular Calcium
Waves in Brain Slices
Trent A.
Basarsky1,
Steven N.
Duffy1,
R. David
Andrew2, and
Brian A.
MacVicar1
1 Department of Physiology and Biophysics, University
of Calgary, Calgary, Alberta Canada T2N 4N1, and
2 Department of Anatomy and Cell Biology, Queens
University, Kingston, Ontario Canada K7L 3N6
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ABSTRACT |
Spreading depression (SD) was analyzed in hippocampal and
neocortical brain slices by imaging intrinsic optical signals in combination with either simultaneous electrophysiological recordings or
imaging of intracellular calcium dynamics. The goal was to determine
the roles of intracellular calcium
(Ca2+int) waves in the generation
and propagation of SD. Imaging of intrinsic optical signals in the
hippocampus showed that ouabain consistently induced SD, which
characteristically started in the CA1 region, propagated at 15-35
µm/sec, and traversed across the hippocampal fissure to the dentate
gyrus. In the dendritic regions of both CA1 and the dentate gyrus, SD
caused a transient increase in light transmittance, characterized by
both a rapid onset and a rapid recovery. In contrast, in the cell body
regions the transmittance increase was prolonged. Simultaneous imaging
of intracellular calcium and intrinsic optical signals revealed that a
slow Ca2+int increase preceded any
change in transmittance. Additionally, a wave of increased
Ca2+int typically propagated many
seconds ahead of the change in transmittance. These calcium increases
were also observed in individual astrocytes injected with calcium
orange, indicating that Ca2+int waves
were normally associated with SD. However, when hippocampal slices were
incubated in calcium-free/EGTA external solutions, SD was still
observed, although Ca2+int waves were
completely abolished. Under these conditions SD had a comparable peak
increase in transmittance but a slower onset and a faster recovery.
These results demonstrate that although there are calcium dynamics
associated with SD, these increases are not necessary for the
initiation or propagation of spreading depression.
Key words:
calcium waves; astrocytes; spreading depression; intrinsic optical signals; ouabain; hippocampus; migraine
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INTRODUCTION |
Spreading depression (SD) was first
described in cortical tissue >50 years ago (Leao, 1944 ). SD is
classically characterized as a slowly propagating wave of neuronal and
astrocytic depolarization that results in a transient depression of
synaptic transmission (Nicholson and Kraig, 1981; Somjen et al., 1992).
SD propagates radially at 30-50 µm/sec from its initiation site and
is associated with a redistribution of ions, shrinkage of the
extracellular space, and an increase in energy metabolism (Nicholson
and Kraig, 1981). Typically cells recover and are relatively unchanged
after SD. However, ischemic insult can induce SD waves (Nedergaard and Hansen, 1993; Dietrich et al., 1994), and the extent of the resultant damage is increased with each incidence of SD (Mies et al., 1993). The
underlying mechanisms of SD are still unknown.
Recent observations of the interactions between astrocytes and neurons
in cell culture have suggested a novel explanation for the mechanism of
SD initiation and propagation. Astrocytes exhibit waves of increased
intracellular calcium that can propagate through gap junction-coupled
networks of astrocytes. Several laboratories have shown that an
increase in intracellular calcium in cultured glial cells can trigger
an increase in intracellular calcium in neighboring neurons (Charles,
1994; Nedergaard, 1994; Parpura et al., 1994; Hassinger et al., 1995).
These results suggest that complex and dynamic interactions can occur
between glia and neurons. Furthermore, astrocytic calcium waves have
many characteristics that are similar to SD (Nedergaard et al., 1995).
For example, SD propagates at approximately the same rate as astrocytic
calcium waves (Hansen, 1985; Cornell Bell et al., 1990), and both SD
and astrocytic calcium waves appear to depend on functional gap
junction communication (Finkbeiner, 1992; Martins-Ferreira and Ribeiro, 1995; Nedergaard et al., 1995). This has led to the hypothesis that
astrocytic calcium waves play an integral role in SD (Cornell Bell and
Finkbeiner, 1991; Nedergaard, 1994).
To address the role of intracellular calcium waves in the
initiation and propagation of SD, we have examined SD in hippocampal and neocortical slices using a combination of electrophysiology, intrinsic optical imaging, and fluorescence microscopy. Imaging of
intrinsic optical signals provides an excellent approach for examining
SD because the large changes in light transmittance in brain slices
caused by SD are easily detected, and the ability to visualize SD in an
entire brain slice allows for the spatial and temporal mapping of SD
propagation. We have designed a system for the simultaneous imaging of
both intrinsic optical signals and
Ca2+int dynamics. In the following study
we have quantified the spatiotemporal intrinsic optical,
electrophysiological, and Ca2+int events
that occur during SD. We demonstrate that slow
Ca2+int elevations precede a robust
calcium wave that occurs concomitantly with SD. However, these calcium
dynamics can be abolished by the removal of external calcium, without
blocking SD. Therefore, although calcium waves are associated with SD,
it is unlikely that a calcium wave in glial cells is critical for the
successful initiation or propagation of SD.
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MATERIALS AND METHODS |
Slices. Hippocampal or neocortical slices (400 µm)
were prepared from 15- to 25-d-old Sprague Dawley rats. Slices were
maintained in artificial CSF (aCSF) aerated with 95% O2 and 5%
CO2 for a minimum of 1 hr after preparation before
experiments were performed. For calcium-imaging experiments, slices
were placed on filter paper suspended over a solution of continuously
stirring 10 µM fura-2 AM and aCSF in an enclosed
interface chamber that was saturated with a humidified 95%
O2 and 5% CO2 environment at a temperature of
25-30°C. To increase fura-2 AM loading efficiency, the aCSF was
supplemented with an additional 10 mM glucose. The slices were maintained in fura-2 AM and aCSF for 2.5 hr and then transferred to room temperature (20-23°C) aCSF aerated with 95% O2
and 5% CO2 until used. For all experiments the slices were
transferred to a superfusion chamber mounted to the imaging setup
described below. Slices were maintained at 33-34°C and held in place
with platinum wires during the experiment. In experiments with
zero-calcium aCSF (0-Ca2+ aCSF), slices were loaded
with fura-2 AM in regular calcium aCSF as described above. Experiments
were alternately performed on slices that were either in
0-Ca2+ aCSF or regular calcium aCSF. We only used
data from slices in 0-Ca2+ aCSF when the matched
controls showed robust Ca2+ waves in regular calcium
aCSF. This ensured that any lack of a response in a
0-Ca2+ aCSF was not attributable to poor slice
quality. We also ensured that there was adequate dye-loading of cells
in each slice that was used. Before the onset of an experiment, slices
were examined at both 365 and 380 nm fura-2 excitation to ensure that
robust fura-2 dye-loading had occurred. This was evident by the easy identification of individually labeled fluorescent cell bodies. Any
slice that did not show robust loading was discarded.
Imaging. Two independent imaging systems were used to
simultaneously acquire intrinsic optical signals and fura-2
fluorescence signals (Fig. 1). The
intrinsic optical system was composed of a charge-coupled device
(CCD; model 4982, COHU) camera connected to an 8-bit frame grabber
(model DT3155; Data Translations) that was driven by Axon Imaging
Workbench (AIW, version 2.1; Axon Instruments, Foster City, CA). The
illumination source was a standard Zeiss tungsten bulb whose output was
directed through a 750DF20 discriminating filter. The fura-2 system
used an image intensifier (VideoScope model VS-2525) coupled to a CCD
camera (model COHU 4982) that was connected to an Axon Image Lightning
2000 frame grabber operating in 8-bit mode and driven by Axon Imaging
Workbench. A lambda-10 filter wheel (Sutter Instruments) equipped with
model 365HT15 and 380HT15 filters was used to provide alternating
excitation for ratiometric fura-2 measurements. To maintain temporal
synchronization between these two systems, a "master-slave"
configuration was established using transistor-transistor logic (TTL)
pulses generated by AIW and transmitted over the parallel ports of each
computer. Typically four frame averages were acquired. This approach
allowed the visualization of SD at a sampling frequency of 1 Hz, which was sufficiently fast given the relatively slow propagation rate of
SD.
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Figure 1.
Schematic of the acquisition setup. Independent
acquisition systems permitted simultaneous imaging of the intrinsic
optical signals and fura-2-based intracellular calcium dynamics, as
well as extracellular field potentials. Slices were continuously
transilluminated with 750 nm light to monitor intrinsic optical signals
and alternately epi-illuminated by 365 or 380 nm light when calcium
images were acquired. The short-pass (<650 nm) dichroic and short-pass
(<630 nm) barrier filter ensured that the intensified CCD only
detected light from the fura-2 signal. An additional long-pass (>650
nm) filter on the intrinsic optical acquisition system ensured that
there was no crossover of the fura-2 signal into the intrinsic optical
channel. In some cases, extracellular field potentials were recorded.
Typically the intrinsic optical computer provided the synchronizing
trigger outputs to ensure temporal synchrony with the rest of the
acquisition.
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The optical path shown in Figure 1 demonstrates how the intrinsic
optical signal was isolated from the fura-2 fluorescence signal. A 650 nm short-pass dichroic mirror reflected longer wavelengths to the
intrinsic optical detection system and passed the shorter wavelengths
to the fura-2 detection system in which an additional 630 nm short-pass
barrier filter ensured no optical crossover from the intrinsic optical
signals. The intrinsic optical signals were recorded and presented as
subtracted images, with the first image acquired during acquisition
serving as the reference image, which was then subtracted from all
subsequent images during acquisition. The intrinsic optical signals
were acquired at a frequency of 1 Hz but saved to disk at a variable
frequency of between 0.008 and 1.0 Hz to reduce data storage
requirements. Ratiometric images were initially acquired at 0.016 Hz,
and this rate was increased to 1 Hz before the onset of SD. This
configuration allowed for the continuous monitoring of changes in the
intrinsic optical signal, yet minimized photobleaching and
phototoxicity because synchronous ratiometric images were acquired at a
higher rate only when necessary.
One caveat of using these two independent imaging systems was that the
two images in the separate systems were not precisely aligned because
of differences in the light path and detection systems. However, this
was compensated for during analysis by aligning given regions of
interest before intensity measurements were made. To achieve this
alignment, an image that contained three easily identifiable reference
points on the imaging coverslip was acquired on both imaging systems.
Using these three reference points, custom software was used to
mathematically zoom, rotate, and translate the coordinates of the
individual regions of interest on the intrinsic optical signal into the
appropriate coordinates for the fura-2 images.
In some experiments astrocytes were intracellularly injected with the
long wavelength calcium indicator calcium orange, according to the
methods described by Duffy and MacVicar (1995). It should be noted that
when using either fura-2 or Calcium Orange, calcium measurements can
only be obtained from cells close to the surface of the slice
(~75-100 µm deep) because of poor penetration of light at these
excitation wavelengths. In contrast, intrinsic optical signals at 750 nm are sensitive to changes throughout the entire slice.
Electrophysiology. Standard extracellular recording
techniques were used to measure the direct current field potentials
during spreading depression. Microelectrodes were fabricated from 1.5 mm outer diameter borosilicate glass and typically had resistances of
15-20 M when filled with 3 M NaCl. Potentials were
measured with a dual channel NeuroData intracellular amplifier (model
IR-283). To reduce noise, a differential recording configuration
was used with one electrode in the bath and a second electrode embedded into the slice, and the preparation was grounded through a World Precision Instruments (Sarasota, FL) reference electrode (model DRIREF-2SH). Both electrode signals were fed into a Frequency Devices
model 901F Bessel filter unit to generate a third differential signal
that was filtered at 1 kHz. Both unfiltered electrode potentials and
the filtered differential signal were then digitized at 16.67 Hz using
Axoscope (version 1.1, Axon Instruments). To ensure spatial synchrony
between the field potential recordings and the imaging measurements, a
single frame was acquired at the end of each experiment to reveal the
precise location of the electrode. This location was then used to
define a region that was used for measurements of the acquired
intrinsic optical signals. Temporal synchrony for the simultaneous
electrophysiology and imaging was maintained with TTL triggers from the
imaging system driving the electrophysiological acquisition.
Solutions. Regular aCSF contained (in mM) NaCl,
124; KCl, 5; MgCl2, 1.3; CaCl2,
2; glucose, 10; and NaHCO3, 26.2. For the zero-calcium aCSF, calcium was replaced with magnesium and 2 mM EGTA was added, yielding a 0-Ca2+
aCSF that contained (in mM) NaCl, 114; KCl, 5;
MgCl2, 3.3; glucose, 10; NaHCO3,
26.2, and EGTA, 2. The pH was adjusted to 7.37 for both of these
solutions. For extracellular recordings the microelectrode was filled
with 3 M NaCl. A stock solution of 10 mM fura-2
AM was prepared daily by sonication in a solution of pluronic F-127 acid (25% w/w), and DMSO and was used at a final fura-2 AM
concentration of 10 µM.
Statistics. Unless otherwise stated, all statistics were
performed with the Mann-Whitney U test. GB-STAT version
3.53 (Dynamic Microsystems) was used for all statistical
calculations.
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RESULTS |
Imaging hippocampal spreading depression
Spreading depression was initiated by bath application of 100 µM ouabain, dihydro-ouabain, elevations in external
potassium, temperature elevations, or through electrical stimulation.
The most consistent stimulus for induction of SD was ouabain, and all
results presented are from ouabain-induced SD. Figure
2A illustrates the time
course of changes in the intrinsic optical signal during a typical
ouabain-induced SD. An increase in the intrinsic optical signal,
measured as a change in transmittance ( T), represents an increase in
tissue volume, or cell swelling (Holthoff and Witte, 1996). Therefore,
SD is detected optically as a result of dynamic changes in cellular
volume. Spreading depression typically initiated in area CA1 and
propagated throughout this region at a velocity of 16.2 ± 2.9 µm/sec (n = 7, mean ± SEM) and through the
dentate gyrus at 19.3 ± 2.4 µm/sec (n = 8)
(Fig. 2). Changes in the extracellular field potential were consistent
with classical SD (Nicholson and Kraig, 1981) and were tightly
correlated with changes in the intrinsic optical signal (Fig.
2C). In the hippocampus, the increase in transmittance in
the stratum radiatum of CA1 and the molecular layer of the dentate
gyrus was transient, whereas stratum pyramidale of the CA1 and the
granule cell layer of the dentate gyrus showed a sustained
increase.
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Figure 2.
Imaging spreading depression in the hippocampus.
A, Intrinsic optical signals during superfusion of
regular ASCF containing 100 µM ouabain. Unless otherwise
noted, ouabain addition was started at 1 min 30 sec in all experiments.
Note the initial transient increase in stratum radiatum of CA1 that
propagates throughout the entire slice. B, Demarcation
of the areas that were used for measurements. C,
D, Time course of the intrinsic optical signal and
extracellular field potential. C, Responses in the
stratum radiatum (zone 1) and stratum pyramidale (zone 2) of CA1.
D, Responses in the molecular layer (zone 3) and granule
cell layer (zone 4) of the dentate gyrus. The time course of the field
potential in C resembles the time course of the
intrinsic optical signal in the stratum radiatum. The dashed
lines represent the peaks of the intrinsic optical signal and
the field potential. The pseudocolor bar is a linear
representation of the change in transmittance. Note that the time is
given in minutes and seconds. Scale bars: A, 400 µm;
B, 200 µm.
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Typically SD originated in only one location, although there were a few
slices in which SD initiated in multiple areas of CA1. Any slices with
multiple initiation sites were excluded from our analysis. SD
propagated radially from the initiation site and eventually crossed the
hippocampal fissure to propagate throughout the dentate gyrus. The time
course of the intrinsic optical signal in the dentate gyrus was very
similar to that seen in CA1 (Fig. 2C,D). It was
very rare to see SD initiate in areas outside of CA1. Furthermore, the
wave typically slowed when entering area CA3, and the time course of
recovery of the intrinsic optical signal was delayed in CA3. The
features of SD in CA3 were not investigated further.
Quantification of spreading depression
To analyze such a wave-like event, it was necessary to define
several features of the intrinsic optical signal (Fig.
3). Several regions of interest were
identified on each image. For each given region of interest the time
course of the intrinsic optical signal was measured. To analyze the
kinetics of the onset of SD, the time of peak, time to 20 or 40% of
the peak, the slope between 20 and 40% of the peak, and the maximum
slope between two measurements during the rising phase were determined.
The kinetics of the recovery phase during SD was quantified by
determining the extent of recovery toward initial transmittance levels
during a fixed time period after the peak (30 sec, 1 min, or 2 min), and by measuring the time required to decay from the peak
by a fixed percentage of the peak (25 or 50%). To measure the
propagation velocity, three regions of interest were identified in
either CA1 or the dentate gyrus. The time differential between the
onset of SD in each of these zones was determined, and the distance
between the zones was measured, enabling one to compute the velocity of
the wave between any two regions of interest. The onset of the SD event was defined as the time to 20% of the peak amplitude because this point gave the most consistent velocity measurements. To alleviate any
problems caused by an initial offset in the intrinsic optical signal,
all values were baseline subtracted before any computations were
performed.
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Figure 3.
Quantification of the intrinsic optical signal.
Time course of the intrinsic optical signal from a single zone in the
stratum radiatum of CA1. The onset of an event was defined by the time
to reach 20% of the peak. The onset kinetics were characterized by
determining the slope between 20 and 40% of the peak response, as well
as the maximum slope during the onset phase. The recovery phase was
defined by measuring the percentage decay at fixed time points (30 sec,
1 min, 2 min) after the peak response and by the time taken to decay a
fixed percentage from the peak.
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Cortical spreading depression
In addition to examining SD in hippocampal slices, SD was induced
by ouabain in neocortical slices (Fig.
4). Spreading depression propagation was
measured in two regions that were defined as the outermost one-third
and innermost one-third of the distance from the neocortical surface to
the white matter. At the outermost layer, cortical SD propagated at
33.8 ± 1.9 µm/sec (n = 5, SEM), whereas
propagation was significantly slower in the innermost layer of the
cortex (27.5 ± 2.4 µm/sec; p < 0.05, Mann-Whitney U test). These values are comparable but
slightly faster than the velocities determined in hippocampal slices.
The SD response was observed as a uniform wave propagating throughout
the entire slice with all regions demonstrating a transient increase in
cell swelling, in contrast to the regional differences observed between stratum radiatum and stratum pyramidale of the hippocampus. In general,
the kinetics of the responses were similar between hippocampal and
neocortical slices (Fig. 4C)
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Figure 4.
Imaging spreading depression in neocortical
slices. A, Intrinsic optical signals during superfusion
of aCSF containing 100 µM ouabain. In this case, SD was
initiated at the leftmost visible portion of the slice and propagated
uniformly across the entire slice. B, Description of the
areas that were used for measurements. C, Time course of
the intrinsic optical signal and extracellular field potential for one
region. Note the similar time course of the field potential and
associated intrinsic optical signal. The dashed lines
represent the peaks of the intrinsic optical signal and the field
potential. Scale bars: A, 400 µm; B,
200 µm.
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Intracellular calcium dynamics during spreading depression
To determine what role intracellular calcium waves play in the
initiation and propagation of SD, we examined intracellular calcium
dynamics during SD (Fig. 5). To increase
spatial resolution we used a higher power objective (25× compared with
5×), and limited our imaging to the infrapyramidal blade (free blade)
of the dentate gyrus. A number of technical reasons favored this
imaging location: (1) the dentate gyrus demonstrated the most robust
loading of fura-2 AM; (2) SD always propagated through the dentate
gyrus from the suprapyramidal blade (enclosed blade) into the
infrapyramidal blade, and thus, the direction of wave propagation was
known in advance; and (3) because SD propagated into the dentate gyrus, complications caused by multiple initiation sites were avoided. As
shown in Figure 2D the intrinsic optical signal
response in the dentate gyrus was comparable to the response seen in
CA1.
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Figure 5.
Simultaneous imaging of intracellular calcium and
intrinsic optical signals in the dentate gyrus. A,
Intrinsic optical signals (top) and intracellular
calcium (bottom) during superfusion of aCSF containing
100 µM ouabain. A slow rise in calcium precedes any
detectable change in the intrinsic optical signal, and a calcium wave
preceded the intrinsic optical signal wave. The white
lines denote the regions corresponding to the cell body layer
in the dentate gyrus. The white dots denote the areas
that were used for measurements. B, Because a higher
power objective was required for enhanced spatial resolution, only a
portion of the dentate gyrus was imaged (see Results for details).
C, Time course of the intrinsic optical signal and
intracellular calcium levels during SD. The same data with an expanded
time scale are shown in the bottom panel. The
dashed lines represent the peaks of the intrinsic
optical signal and the calcium ratio. Scale bar in A, 50 µm for both intrinsic and calcium images.
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Using two independent imaging systems it was not possible to ensure
complete spatial registry of the individual images between the two
setups. However, it was possible to ensure spatial registry on given
regions of interest before measurements were made (see Materials and
Methods). This ensured that the extracted intrinsic and calcium
measurements originated from precisely the same slice location.
Coupling this with the synchronizing triggers between the two systems
ensured tight spatial and temporal synchrony for analysis purposes. It
must be noted that for display purposes, the raw images were not
translated, and instead, the regions corresponding to the cell body
layer in the dentate gyrus are outlined on both the intrinsic optical
and fura-2 signals (Fig. 5A, white
lines).
In all cases, an increase in intracellular calcium was detected during
SD (n = 14). Typically, a slow calcium rise preceded any increase in the intrinsic optical signal and, subsequently, a
calcium wave propagated through the dentate gyrus, ahead of the
intrinsic optical signal (Fig.
5A,C) Because the calcium signal was composed of a slow rise and a subsequent wave, three different onset parameters were used to demonstrate that the calcium signal preceded the intrinsic optical signal (Fig.
6). The calcium signal peaked 15.8 ± 2.2 sec ahead of the intrinsic optical signal and propagated through
the slice at a velocity of 21.1 ± 6.3 µm/sec. In comparison,
the propagation velocity of the associated intrinsic optical signal was
24.8 ± 2.7 µm/sec, comparable to the velocity of 19.3 ± 2.4 µm/sec that had been measured at lower magnification (p > 0.05, Mann-Whitney U test). On
occasion only a slow elevation in calcium without the calcium wave was
observed, or only a wave occurred without the slow rise.
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Figure 6.
Intracellular calcium elevations preceded the
onset of the intrinsic optical signal. The time differential between
intracellular calcium elevations and the intrinsic optical signal is
shown. The  time is given for three different onset parameters, 20%
of the peak, 40% of the peak, and the time of the peak. These three
parameters are described in detail in Figure 3. In all measurements the
calcium signal preceded the intrinsic optical signal.
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It has been established that calcium waves occur in cultured astrocytes
(Cornell Bell et al., 1990). To determine whether glia cells
contributed to the observed Ca2+int
dynamics, astrocytes were injected intracellularly with the calcium
indicator calcium orange (Fig. 7). Under
these conditions, any detectable change in
Ca2+int would be attributable to
astrocytic Ca2+int dynamics. Associated
with spreading depression, an increase in astrocytic
Ca2+int was observed, and the peak of
these increases preceded the intrinsic optical signal of SD
(n = 4). These results demonstrate that an increase in
Ca2+int precedes spreading depression
and suggest that a calcium wave is involved in the initiation or
propagation of spreading depression. Furthermore, such calcium dynamics
are attributable in part to astrocytic contributions.
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Figure 7.
Imaging of astrocytic intracellular calcium and
intrinsic optical signals in the hippocampus. A,
Intrinsic optical signals (top) and intracellular
calcium (bottom) during bath application of 100 µM ouabain, which was started at 2 min. Individual
astrocytes were intracellularly injected with calcium orange. The
dotted lines represent the onset of the calcium signal,
the peak of the calcium signal, and the peak of the intrinsic optical
signal. Note that the peak of the calcium signal precedes the peak of
the intrinsic optical signal.
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Spreading depression in the absence of external calcium
To determine whether the SD-associated increases in
Ca2+int are necessary for the initiation
and propagation of SD, we evoked SD in the absence of external calcium.
After incubation of the slices in 0-Ca2+ aCSF with 2 mM EGTA (0-Ca2+ aCSF) for >20 min, SD
initiation and propagation was still induced by superfusion of ouabain
(Fig.
8A,C).
However, Ca2+int increases during SD
were completely inhibited (Fig.
8D,E). In regular calcium aCSF, the
baseline fura-2 ratio was 0.77 ± 0.02 and peaked at 1.12 ± 0.10 (p < 0.001, Student's t test)
during SD. In 0-Ca2+ aCSF, the baseline was
0.73 ± 0.01 and during SD reached 0.76 ± 0.01 (p > 0.05, Student's t test). The
inhibition of Ca2+int dynamics without a
concomitant inhibition of SD indicates that the increase in
Ca2+int observed in regular aCSF is not
critical for the initiation or propagation of SD.
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Figure 8.
Spreading depression in the absence of external
calcium. A, Intrinsic optical signals in
0-Ca2+ aCSF during bath application of 100 µM ouabain. B, Description of the areas
that were used for measurements. C, Time course of the
intrinsic optical signal in stratum radiatum and stratum pyramidale of
CA1. D, Intracellular calcium increases are absent in
0-Ca2+ aCSF. Left, Before spreading
depression. Right, Peak of spreading depression. The
white dots denote the areas of measurement shown in
E. E, Time course of the intrinsic
optical signal and intracellular calcium levels in
0-Ca2+ aCSF. Note the complete absence of any
calcium increase, although SD still occurred. Scale bars:
A, 400 µm; B, 200 µm;
D, 50 µm (top and
bottom).
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The peak amplitudes of the intrinsic optical signal were similar in
regular and 0-Ca2+ aCSF (Fig.
9A), indicating that the
extent of swelling was comparable in both conditions. However, SD in
0-Ca2+ aCSF was not identical to SD in regular aCSF.
In 0-Ca2+ aCSF, SD had a smaller onset slope and
lower maximum onset slope, indicative of a slower rise time (Fig.
9B,C). In contrast, the kinetics of
recovery were faster in 0-Ca2+ aCSF, as shown by the
faster rate of decay and decreased time to a fixed decay percentage
(Fig. 9D,E).
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Figure 9.
Analysis of spreading depression in the presence
and absence of external calcium. The amplitudes of the peak response
are shown in A. Note that the peak responses are
expressed as T/T, where T was defined as the initial transmittance
level for each zone. The rising phase of SD was decreased in the
absence of calcium. The onset slope, measured as either the slope
between 20 and 40% of the peak (B), or the
maximum slope from onset to peak (C), was smaller
in the absence of calcium. The recovery phase is faster in
0-Ca2+ aCSF (D, E).
The time to decay 25 or 50% from the peak is shown in
D, whereas the rate of decay defined as the percentage
decay over a fixed time interval is given in E.
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DISCUSSION |
We have used a combination of imaging and electrophysiological
techniques to record the propagation of SD in the rat hippocampus. Imaging of intrinsic optical signals permitted us to map the
propagating wave front and to correlate it with the
Ca2+int changes monitored with fura-2
fluorescence. In normal solutions, SD was associated with increases in
Ca2+int that had a wave-like appearance,
and part of this Ca2+int increase was of
astrocytic origin. However, calcium waves were not critical for SD,
because SD was still observed in the absence of external calcium and
any detectable increase in Ca2+int.
Intrinsic optical signals
These experiments demonstrate that imaging intrinsic optical
signals can be used to monitor slow activity-dependent changes in brain
tissue and to provide novel insights into the cellular mechanisms of
SD. Intrinsic optical signals develop over seconds (MacVicar and
Hochman, 1991) and reflect the cellular changes in volume that occur in
response to activity (Andrew and MacVicar, 1994). Changes in light
transmittance have been shown to be directly correlated with
extracellular space changes (Holthoff and Witte, 1996). Cellular
swelling causes a decrease in extracellular space and an increase in
light transmittance, whereas cell shrinkage causes the reverse. It
should be noted that the slices we imaged were composed of both neurons
and glia and that the intrinsic optical signal could represent volume
changes in both cell types.
The maintained light transmittance increase in the cell body region
versus the transient increase in the dendritic region during SD may
reflect different regional and cellular patterns of volume changes. The
maintained increase in stratum pyramidale during SD likely indicates
that neurons underwent swelling. Previous reports have shown that
neurons swell (Van Harreveld and Khattab, 1967) and that the
extracellular space shrinks during SD (Nicholson and Kraig, 1981; Jing
et al., 1994), consistent with our observations. The transient increase
in light transmittance in stratum radiatum is more difficult to
interpret. A previous study suggested that the increase in light
transmittance in hippocampal slices during synaptic activity results
from K+ accumulation and swelling in astrocytes
(MacVicar and Hochman, 1991). It is possible that the subsequent
decreases in the dendritic region reflect compensatory astrocytic
regulatory volume decreases. Regulatory volume decrease has been
observed in astrocytes in cell culture (Pasantes-Morales et al., 1994)
but not in the CNS to date (Krizaj et al., 1996; Andrew et al., 1997).
Alternatively, the decrease in light transmittance in the dendritic
region could represent neuronal dendritic shrinkage. In support, it
recently has been shown that neurons are capable of undergoing
regulatory volume decrease (Churchwell et al., 1996), although this was
under conditions in which NMDA receptors were inhibited. Therefore, our
study illustrates the utility of imaging intrinsic optical signals in
mapping SD. Further studies are in progress to differentiate the
underlying neuronal and astrocytic cellular changes.
Ouabain induction of spreading depression
Although it has long been known that ouabain can induce SD by
inhibiting Na+,K+ ATPases, the
sequence of events that leads to SD is unknown (Nicholson and Kraig,
1981; Haglund and Schwartzkroin, 1990). At the concentrations that we
used ouabain will inhibit both the  2 and 3 isoforms of
Na+,K+ ATPase that are thought to
be expressed mainly in astrocytes and neurons, respectively (Watts et
al., 1991; Sweadner, 1992). Inhibiting
Na+,K+ ATPases would increase
intracellular Na+ and extracellular
K+ concentrations, leading to cellular
depolarization, which could contribute to the triggering of SD. It has
been suggested that the sensitivity of the CA1 region to the induction
of SD by ouabain is attributable to the reduced amounts of
Na+,K+ ATPases in CA1 compared
with CA3 (Haglund et al., 1985; Haglund and Schwartzkroin, 1990). Our
observation that SD was typically initiated in the CA1 region
is consistent with this.
Although ouabain can induce cell death (Lees et al., 1990; Lees, 1991)
our observations of fura-2-loaded cells suggest that this was not a
significant problem during the time course of our experiments. We did
not observe maintained calcium elevations that typically indicate cell
death. Furthermore, we did not observe any decrease in the intensity of
fura-2 fluorescence at the near isosbestic wavelength of 365 nm that
would occur if there was any dye loss because of cell lysis.
Astrocyte calcium waves and spreading depression
Our studies were designed to test the hypothesis that
Ca2+int waves in glial cells are
important in SD (Cornell Bell and Finkbeiner, 1991; Nedergaard, 1994).
There is increasing evidence that astrocytes and neurons are capable of
bidirectional communication. For example, it has been shown that
neuronal activity can induce elevations in
Ca2+int in neighboring astrocytes (Dani
et al., 1992). Conversely, there is evidence that elevations in
Ca2+int in astrocytes can induce both
neuronal depolarization and elevations of neuronal
Ca2+int (Charles, 1994; Nedergaard,
1994; Parpura et al., 1994; Hassinger et al., 1995). Because SD is
characterized as a slowly propagating wave of neuronal and glial
depolarization, it has been postulated that such complex interactions
between neurons and glia are necessary for SD. In fact, several
features of SD and astrocytic calcium waves, such as velocity of
propagation (Hansen, 1985; Cornell Bell et al., 1990) and sensitivity
to gap junction inhibitors, (Finkbeiner, 1992; Martins-Ferreira and
Ribeiro, 1995; Nedergaard et al., 1995) are very similar (Nedergaard et
al., 1995). Therefore, it has been proposed that astrocytic calcium
waves play a role in the initiation and propagation of SD (Cornell Bell
and Finkbeiner, 1991; Nedergaard, 1994).
How might an astrocytic calcium wave be involved in spreading
depression? It has been demonstrated that elevations in astrocytic calcium result in the subsequent depolarization and elevation of
Ca2+int in neighboring neurons (Charles,
1994; Nedergaard, 1994; Parpura et al., 1994; Hassinger et al., 1995).
Two mechanisms have been proposed to explain such glia-neuron
signaling. Elevations of Ca2+int in
astrocytes causes the release of glutamate, which depolarizes adjacent
neurons and activates neuronal NMDA receptors (Parpura et al., 1994;
Hassinger et al., 1995). Astrocytes have also been postulated to be
transiently coupled to neurons through gap junctions, allowing for the
propagation of a calcium wave between the two cell types (Nedergaard,
1994). Both of these mechanisms are consistent with the ability of a
propagating astrocytic calcium wave to induce propagating neuronal
depolarization, similar to that which is observed in SD. Such a
mechanism would imply that Ca2+int
changes should precede SD. We typically observed slow
Ca2+int elevations minutes before the
onset of SD, and a calcium wave typically preceded SD by many seconds.
Experiments performed with individual astrocytes loaded with fura-2
revealed that astrocytic Ca2+int changes
partially account for the observed
Ca2+int dynamics. Taken together, these
data are consistent with the idea that astrocytic calcium waves are
normally associated with SD.
Zero-calcium and spreading depression
To directly test the involvement of calcium waves in SD, we
initiated SD in slices incubated in 0-Ca2+ aCSF. In
regular aCSF the calcium waves that we observed are most likely
attributable to calcium influx or calcium release from internal calcium
stores. Therefore, we expected that the magnitude of the calcium wave
would be smaller in 0-Ca2+ aCSF. For example,
calcium waves in astrocytes in cell culture and in retinal glia (Newman
and Zahs, 1997) are primarily attributable to IP3-mediated
release of calcium from intracellular stores (Charles et al., 1993),
and these waves exist in the absence of external calcium (Newman and
Zahs, 1997). Our observations that there was no detectable increase in
[Ca2+]int during SD in
0-Ca2+ aCSF suggests that release from internal
stores does not contribute to the calcium waves we observed in SD.
However, preincubating slices for 20 min in 0-Ca2+
aCSF could have depleted calcium stores before SD was initiated, thus
accounting for the lack of any calcium signals generated by release
from internal stores. In agreement, Venance et al. (1997) have
demonstrated that internal calcium stores in cultured astrocytes are
depleted within 10 min after incubation in a 0-Ca and 2 mM
EGTA external solution. An alternative explanation for the lack of any
detectable calcium dynamic during SD in 0-Ca2+ aCSF
is that the calcium increase is attributable to influx through voltage-gated calcium channels. In addition to neuronal calcium channels, it has been reported that astrocytic calcium channels are
activated when extracellular K+ exceeds 25 mM (Duffy and MacVicar, 1994) within the 25-80
mM range reported during SD (Vyskocil et al., 1972). Our
observation that SD still occurred in the absence of any detectable
Ca2+int response is consistent with
previous reports that SD in the hippocampus is not inhibited by calcium
channel antagonists (Jing et al., 1993; Herreras et al., 1994).
However, it should be noted that in the retina, SD is inhibited in the
absence of external calcium (Martins-Ferreira et al., 1974; Nedergaard
et al., 1995).
Taken together, these data demonstrate that although calcium elevations
are associated with SD, such calcium dynamics are not critical for SD,
suggesting that SD propagation can occur by a mechanism independent of
calcium wave propagation. Our results demonstrate that SD can occur in
the hippocampus with no detectable increase in
Ca2+int. The underlying mechanisms of SD
remain to be determined.
| |
FOOTNOTES |
Received Feb. 2, 1998; revised May 21, 1998; accepted June 24, 1998.
This work was supported by the Medical Research Council (MRC). We thank
J. Armstrong, D. Doll, and V. Parpura for helpful comments on this
manuscript. We also thank A. Salkauskas and S. Borg for helpful
discussions on analysis software development. T.A.B. is a postdoctoral
fellow of the Alberta Heritage Foundation for Medical Research (AHFMR).
B.A.M. is an MRC Senior Scientist and an AHFMR Scientist.
Correspondence should be addressed to Dr. Brian MacVicar, Department of
Physiology and Biophysics, University of Calgary, 3330 Hospital Drive
NW, Calgary, Alberta Canada T2N 4N1.
| |
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B. Innocenti, V. Parpura, and P. G. Haydon
Imaging Extracellular Waves of Glutamate during Calcium Signaling in Cultured Astrocytes
J. Neurosci.,
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[Abstract]
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M. Muller and G. G. Somjen
Intrinsic Optical Signals in Rat Hippocampal Slices During Hypoxia-Induced Spreading Depression-Like Depolarization
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[Abstract]
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F. Blomstrand, C. Giaume, E. Hansson, and L. Ronnback
Distinct pharmacological properties of ET-1 and ET-3 on astroglial gap junctions and Ca2+ signaling
Am J Physiol Cell Physiol,
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[Abstract]
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T. A. Basarsky, D. Feighan, and B. A. MacVicar
Glutamate Release through Volume-Activated Channels during Spreading Depression
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Top
Abstract
Introduction
