Departments of 1Physiology and
2PediatricsConstance Kaufman Research
Laboratory, and 3Neuroscience Program, Tulane
University School of Medicine, New Orleans, Louisiana 70112
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ABSTRACT |
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Kreisman, Norman R., Soheil Soliman, and David Gozal. Regional Differences in Hypoxic Depolarization and Swelling in Hippocampal Slices. J. Neurophysiol. 83: 1031-1038, 2000. Pyramidal neurons in the CA1 region of the hippocampus are highly vulnerable to damage from hypoxia-ischemia, whereas neurons in the CA3 region and the dentate gyrus are more resistant. A similar pattern of vulnerability to loss of synaptic and membrane function occurs in the in vitro hippocampal slice preparation, suggesting that intrinsic factors are important in acute neuronal damage. Simultaneous recordings of DC potential and imaging of changes in light transmittance were made in slices from the middle one-third of the hippocampus to characterize the initiation and spread of depolarization and swelling during hypoxia-aglycemia. Hypoxic depolarization (HD) and associated optical changes were initiated simultaneously in the stratum oriens of the CA1 region and thereafter spread to the stratum radiatum of CA1 and later to the upper (inner) blade of the dentate gyrus. A decrease in light transmittance was associated consistently with depolarization in all regions (n = 22 slices). Investigation of the sequence of activation in intact slices showed that activation of the dentate gyrus arose independently of activation of the CA1 region. This was confirmed by recordings made from minislices in which CA1, CA3, and dentate regions were physically separated. HD and optical changes were never observed in the CA3 region, despite exposure to 40-60 min of combined hypoxia and aglycemia. In contrast, exposure to hypoxia after pretreatment of slices with altered tonicity or ion composition, which triggered episodes of spreading depolarization (SD), provoked depolarization and optical changes simultaneously in both CA1 and CA3 regions. Similarly, pretreatment with agents that cause severe metabolic impairment, such as dinitrophenol (DNP), also rendered the CA3 region vulnerable to subsequent hypoxia. This suggests that the CA3 region in hippocampal slices is normally resistant to HD and only becomes vulnerable after severe impairment of metabolic capacity. The similar order of vulnerability of in vitro and in vivo hippocampus to hypoxia-aglycemia supports the use of the hippocampal slice preparation to investigate early changes potentially contributing to hypoxic-ischemic injury.
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INTRODUCTION |
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Brain injury after ischemia shows a well-known
pattern of regionally selective vulnerability (Kirino and Sano
1984; Pulsinelli et al. 1982
). In the
hippocampal formation, the CA1 region is highly vulnerable to damage
from even brief episodes of ischemia, whereas the CA3 region and the
dentate gyrus are more resistant (Schmidt-Kastner and Freund
1991
). In the in vitro hippocampal slice preparation, a similar
pattern of electrophysiological vulnerability emerges during acute
hypoxia and/or aglycemia, suggesting the importance of intrinsic
factors in acute neuronal damage. Irreversible blockade of synaptic
transmission after acute hypoxia is an important physiological
indicator of injury (Schiff and Somjen 1987
;
Schurr et al. 1989
). Several laboratories have confirmed
regional differences in the susceptibility to irreversible synaptic
blockade, showing that CA1 is highly susceptible compared with the
dentate gyrus or the CA3 region (Aitken and Schiff 1986
;
Crépel et al. 1992
; Kass and Lipton
1986
).
A putative factor contributing to hypoxic injury is the profound
depolarization of neurons, and presumably glia, due to a substantial
increase in membrane permeability triggered by hypoxia and/or
hypoglycemia (Balestrino 1995; Hansen and Zeuthen
1981
; Vysko
il et al. 1972
). The
depolarization, called hypoxic depolarization (HD), occurs
within minutes after hypoxia-induced synaptic blockade. HD is heralded
by a profound negative shift of the extracellular potential, virtually
identical with that seen in spreading depression of
Leão (Aitken et al. 1998
; Somjen et
al. 1990
; Somjen et al. 1992
). In hippocampal
slices, synaptic transmission fails to recover after reoxygenation if
HD lasts more than a few minutes (Balestrino et al.
1989
; Roberts and Sick 1988
) and there is
evidence that HD contributes to acute cellular damage (Obeidat
and Andrew 1998
). Simultaneous recordings of the extracellular
potential in CA1 and dentate gyrus during hypoxia reveal that CA1
always depolarizes before the dentate gyrus (Aitken and Schiff
1986
; Balestrino et al. 1989
). However, the
susceptibility of the CA3 region to HD varies among several reports,
probably because of differences in experimental conditions (e.g., see
Aitken et al. 1998
; Davis et al. 1986
;
Obeidat and Andrew 1998
).
Many of these experiments used recordings of extracellular potential
from one or two microelectrodes to assess the localization of HD.
Unfortunately, this punctate view cannot provide adequate information
about the origin, spread, and spatial extent of HD. Recently, imaging
techniques have been applied to the mapping of hypoxia- or
glutamate-induced depolarization by measuring associated changes in
light transmittance that are ostensibly due to cell swelling
(Aitken et al. 1998; Andrew et al. 1996
;
Kreisman et al. 1996
; Obeidat and Andrew
1998
; Turner et al. 1995
). HD is accompanied by
cellular swelling because the dissipation of ion gradients across the
cell membrane osmotically obligates the influx of water into the cell.
These investigations have corroborated the particular susceptibility of
CA1 to both HD and swelling. Moreover, recent imaging studies have
localized where HD is initiated and have described the spread of HD in
submerged hippocampal slices (Obeidat and Andrew 1998
)
and interfaced hippocampal slices (Kreisman et al.
1996
). In the present investigation we imaged changes in light
transmittance to verify the regional susceptibility to HD in interfaced
slices, as an extension of our earlier preliminary report.
Additionally, we examined whether HD spreads from more susceptible to
less susceptible regions or is initiated independently in each region,
using both intact slices and minislices of individual regions. Also, we
determined whether regions normally resistant to HD could be made
vulnerable to HD by various treatments. Finally, we attempted to
determine whether susceptibility to HD, or lack thereof, might be
related to selective neuronal injury resulting from the preparation of
hippocampal slices.
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METHODS |
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Preparation of hippocampal slices
Male Sprague-Dawley rats (200-500 g; Charles River) were
anesthetized with ether and perfused through the heart with 60 ml of
cold (4°C) bathing medium containing (in mM): 129 NaCl, 3.5 KCl, 2 MgSO4, 1 NaH2PO4, 2.7 CaCl2, 26 NaHCO3, and 10 glucose (according to Kreisman and LaManna 1999). The
rats were decapitated, the brain removed, and the tissue placed
immediately in ice-cold bathing medium. Transverse slices
(400-µm-thick) were cut with a mechanical tissue chopper (Stoelting,
Wood Dale, IL) from the middle one-third of the hippocampus to avoid
septotemporal gradients of excitability (Bragdon et al.
1986
). Slices were then placed in the wells of a holding
chamber on filter paper (Whatman 50) thoroughly wetted with bathing
medium and gased with humidified 95% O2-5%
CO2. The incubation medium was maintained at room
temperature (23-24°C) and was replaced with fresh medium at 45-min
intervals. In some experiments, slices were preincubated at room
temperature in a modified bathing medium containing 400 µM ascorbate,
with zero Ca2+ and 10 mM
Mg2+ substituted for the usual 2.7 mM
Ca2+ and 2 mM Mg2+. After
90-120 min of preincubation, one of the slices was transferred to the
nylon mesh of an interface-style recording chamber containing normal
medium, whereas others remained in the holding chamber. The temperature
in the recording chamber was maintained at 33-34°C and the slice was
subfused with standard bathing medium flowing at 0.6 ml/min. Warmed,
humidified 95% O2-5%
CO2 superfused the slice at a rate of 480 ml/min.
Osmolarity of the standard bathing medium was 295-300 mOsm/l.
Hypotonic media of various strengths were made by removing the
appropriate amount of NaCl.
Electrical stimulation and recording
Viability of the CA1 region was tested by stimulating the
Schaffer collaterals with constant-current pulses (400 µA; 0.2 ms) using glass micropipettes filled with 150 mM NaCl (tip resistance 5-20
M). Only stable recordings of population spikes with a minimum amplitude of 3 mV were acceptable. We attempted to test viability of
the CA3 region by stimulating the fimbria and recording antidromic or
orthodromic population spikes from the nearby CA3 stratum
pyramidale. Although prevolleys were present, orthodromic
population spikes were commonly absent. Thereafter, CA1 and CA3
recording electrodes were usually relocated to the stratum radiatum for
recording extracellular DC potentials. Excitatory postsynaptic
potentials (fEPSPs) also were recorded extracellularly in CA1 s.
radiatum in response to stimulating the Schaffer collaterals with
constant-current pulses (50-100 µA; 1 ms) to produce a response that
was 50-75% of maximum amplitude. Extracellular DC levels and optical
signals (see following section) were recorded continuously on a
strip-chart recorder, and evoked responses and optical signals were
digitally acquired with MacLab software.
Measurement and imaging of light transmittance
Transmitted light from a stable quartz-halogen source was
detected by a silicon photodiode (model 78-7821; Ealing), coupled to
one ocular of a Nikon, SMZ-2 binocular dissecting microscope. The
optical field was approximately 0.4 × 1.2 mm, which included the
s. radiatum and s. pyramidale of the CA1 region. To calibrate the
optical signal, basal light transmittance (T) was nulled at the beginning of the experiment, using the offset of a DC amplifier. The dark value was then determined by shutting off the light source. Variations in light intensity from zero (T) were
calculated as
T/T in percent.
Imaging of light transmittance was accomplished by coupling an 8-bit
digital video camera (model CCD72, Dage MTI, Michigan City, IN) to the
remaining ocular of the binocular dissecting microscope. Black levels
and dynamic range of the video signals were adjusted manually, based on
a histogram of light transmittance values obtained from each slice
during normoxia. As with the photodiode measurements, relative changes
in light transmittance (T) were expressed as a percent of
T. Images were captured at 0.05-3.0 Hz, depending on the
individual experiment, using custom-designed imaging software (Synetic,
Montreal, Quebec, Canada). Difference images were derived by
subtracting the mean of five images at peak hypoxia from the mean of
five images taken during normoxia. Pseudocoloring of the difference
images was accomplished with Adobe Photoshop software. Areas of
interest were manually drawn on an image of the slice, and
T/T was plotted as a function of time for each area.
Induction of hypoxia
Severe hypoxia was induced by switching the gas mixture
superfusing the slices from 95% O2+5%
CO2 to 95% N2+5%
CO2. In several experiments,
PO2 in the bathing medium was
measured polarographically, by using a platinum electrode polarized to
0.7 V relative to an Ag-AgCl wire connected to ground. The mean
PO2 values in the upper 1 mm of the
bath during normoxia and hypoxia were 443 ± 19 mmHg and 20 ± 3 mmHg, respectively (n = 9).
Histology
Slices were incubated in the holding chamber at room temperature
for 2 h and then at 33-34°C for 1 h before fixation
overnight in 10% buffered formaldehyde. This was followed by
incubation in 30% sucrose at 4°C for 24 h. Frozen sections were
cut at 10 µm and mounted on gelatin-coated slides. Sections were
stained with cresyl violet and the morphology of neurons in the CA1 and CA3 pyramidal layers was evaluated and scored according to the scheme
described by Raley-Sussman et al. (1997). Reported
values are the means of ratings made by two observers who were blinded to the treatment the slices received.
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RESULTS |
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Optical and electrical responses to severe hypoxia in various hippocampal regions
Within minutes after onset of severe hypoxia, simultaneous rapid
decreases in light transmittance (plotted here as upward deflections of
T/T) and negative shifts of the extracellular potential (DC), indicative of hypoxic depolarization, occurred in the
CA1 region in all 22 slices tested (see Fig.
1 for example). The optical response
originated in the stratum oriens of the CA1 region and spread laterally
in both directions, also spreading to the s. radiatum. The extent of
lateral spread in CA1 was always greater than the extent of vertical
spread, with a clear demarcation of swollen and normal areas at the
borders of the CA2 region, the subiculum, and the upper blade of the
dentate gyrus. The most intense optical changes always occurred in the
s. radiatum, followed by the s. oriens. In comparison, the
optical responses in the s. pyramidale were minimal (Fig.
1A). After a short delay, the optical response was also
observed in the dendritic layer of the upper blade of the dentate gyrus
in 20 of 22 slices, but in no case did the optical response involve the
lower blade of the dentate gyrus (e.g., Fig. 1B). The mean
latency (±SE) from onset of severe hypoxia to rapid HD and swelling
was 3.0 ± 0.6 min in the CA1 region (n = 22 slices) and 3.7 ± 0.7 min in the upper blade of the dentate gyrus
(n = 20 slices). Similar optical changes were observed
in the subiculum after more prolonged hypoxia, but no attempt was made
to quantify these latencies. Neither HD nor appreciable optical changes
were observed in the CA3 region in these 22 slices, despite extending
the period of severe hypoxia in five experiments (in three cases with
superimposed aglycemia) for more than 40 min (e.g., Fig. 1,
A-C). A summary of optical changes during
various intervals of hypoxia in four regions of hippocampal slices is shown in Table 1.
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The sequence of optical images generated in these experiments suggested
that hypoxic swelling did not spread from the CA1 region to the dentate
gyrus but rather was initiated independently in the two regions (Fig.
1A). To verify this, hippocampal slices were divided into
three minislices by using microscissors to isolate the CA1, CA3, and
dentate regions from one another (Fig.
2A; n = 3 slices). Both pseudocolored difference images (hypoxia minus normoxia;
Fig. 2B) and plots of T/T in the
various minislices as a function of time (Fig. 2C) showed
clearly that swelling occurred independently in the CA1 region and the
upper blade of the dentate gyrus. Identically with intact slices,
virtually no optical changes were observed in either CA3 minislices or
minislices from the lower blade of the dentate gyrus during more than
20 min of severe hypoxia.
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Induction of vulnerability to hypoxic depolarization and swelling in the CA3 region
The results suggested that the CA3 region is more resistant to
hypoxia than CA1, which is in agreement with data obtained in models in
vivo of ischemia (Kirino and Sano 1984;
Pulsinelli et al. 1982
). However, previous reports have
suggested that the preparation of hippocampal slices injures CA3
neurons compared with CA1 neurons (Johnston et al. 1992
;
Newman et al. 1995
; Rice et al. 1994
).
Therefore, we undertook a series of experiments to test whether CA3 was
in fact capable of depolarizing. This was attempted in 26 slices from
an additional 17 rats by using a variety of treatments before and
during induction of hypoxia, which were designed either to increase the
intensity of the metabolic insult (e.g., by adding aglycemia or
treatment with DNP to hypoxia) or to increase the excitability of the
hippocampal slice (Table 2). Hypotonia
was used to increase excitability, often in conjunction with increases
in extracellular [K+] or removal of
extracellular [Ca2+] or
[Mg2+]. Hypotonia swells cells and thereby
decreases interstitial volume, which increases excitability via field
effects (Andrew 1991
; Taylor and Dudek
1984
). For example, strong hypotonia is known to trigger episodes of SD in the CA1 region (Chebabo et al. 1995
),
but it is unknown whether the CA3 region also is susceptible to SD
during hypotonia. Another goal was to make the interstitial volume in CA3 more like that in CA1 to test if that would render CA3 more vulnerable to hypoxic depolarization.
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During normoxia, the treatments described occasionally led to episodes
of SD in CA1 alone (3 of 26 slices) or in both CA1 and CA3 (4 of 26 slices; Table 2). The occurrence of SDs during normoxia predisposed
both the CA1 and CA3 regions to depolarize subsequently
during hypoxia in six of these seven slices (Table 2). The results of
one of these experiments is shown in Fig. 3. Decreasing the osmolarity of the
bathing medium from 300 mOsm/l to 200 mOsm/l caused a positive shift of
7-8 mV in the DC potential in both regions, probably because of the
change in ionic strength of the bathing medium. This was followed in
~5 min by a prolonged negative shift in the baseline DC potential
with superimposed episodes of SD in both the CA1 and CA3 regions. In
this example, the initial SD occurred in CA1 seconds before it occurred
in CA3, but the latency between the two responses decreased with
successive episodes. The fourth episode of SD in this case occurred
simultaneously in both regions. Measurements of
T/T as a function of time also revealed
simultaneous, rapid changes indicative of SD in both CA1 and CA3 (Fig.
4B). Images taken at the
steady-state period between episodes of SD (i.e., at the 15-min mark of
Fig. 4B) showed osmotic swelling primarily in CA1 and patchy
swelling in CA3 (Fig. 4A). Hypoxia was induced 5 min after
the four episodes of SD (Fig. 3). Hypoxic depolarization (HD) was
triggered both in CA1 and CA3 within ~1 min after onset of hypoxia in
the presence of the hypo-osmotic medium. CA3 depolarized before CA1 in
this instance. Induction of hypoxia also produced rapid shifts in the
optical traces, indicative of swelling, in CA1, CA3, and the upper
blade of the dentate gyrus (Fig. 4D). Imaging at 8 min of
hypoxia showed clear swelling of both the entire CA1 and CA3 regions
(Fig. 4C). Similar responses were observed in three slices.
In two other slices, treatment with DNP to deplete energy supplies
resulted in rapid depolarization in both CA1 and CA3 during hypoxia, in one case despite the absence of preceding SDs (Table 2).
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Morphology of CA1 and CA3 pyramidal neurons and neurons of the dentate gyrus
The morphology of neurons in the pyramidal cell layer of the CA1
and CA3 regions was evaluated to determine primarily whether CA3
neurons were preferentially damaged by the slicing procedure, as
reported by others (Newman et al. 1995; Rice et
al. 1994
). Pyramidal neurons were classified in three
categories, based on their appearance under the light microscope
(according to Raley-Sussman et al. 1997
). Class A
neurons had intact, well-defined membranes, a clear uniform nucleus,
distinct nucleoli, and a clear cytoplasm. Class B neurons had an
indistinct nucleus, were darkly stained, and were shrunken and
distorted in shape. Class C neurons had no distinct nuclear boundary
and had either a vesiculated cytoplasm or were obviously swollen.
Examples of the histological appearance of CA1 and CA3 pyramidal
neurons are shown in Fig. 5, A
and B. The percentage of class A neurons in CA3 pyramidale
was lower than in CA1 pyramidale, whereas the percentage of class C
neurons was higher in CA3 than in CA1, but all differences were
relatively small in magnitude (Table 3).
Some CA3 neurons contained vacuoles and had blebbing of their membranes
(Fig. 5B). The tightly packed neurons of the upper blade of
the dentate gyrus were round and had intact membranes and a distinct
nucleolus (Fig. 5C). In contrast, neurons of the lower blade
of the dentate gyrus were smaller than those of the upper blade, and
many were less distinctly stained (Fig. 5D). However, the
neurons of the lower blade were neither pyknotic nor swollen.
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In four experiments, slices were preincubated in a modified bathing
medium in which 400 µM ascorbate was added to the standard medium and
zero Ca2+ and 10 mM Mg2+
substituted for the usual 2.7 mM Ca2+ and 2 mM
Mg2+. This has been shown to result in slices
with improved morphology in CA3 pyramidal neurons (Newman et al.
1995; Rice et al. 1994
). The rationale was that
improved viability of the CA3 neurons might allow them to respond to
hypoxia with HD. HD still failed to occur in the CA3 region of slices
preincubated in this modified medium.
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DISCUSSION |
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Four major findings have emerged from this investigation: 1) CA1 and, to a lesser extent, the upper blade of the dentate gyrus in hippocampal slices are susceptible to hypoxic depolarization, whereas the CA3 region and the lower blade of the dentate gyrus are resistant. 2) The magnitude and time course of hypoxic depolarization and changes in light transmittance indicative of swelling coincide, regardless of the hippocampal region being investigated. 3) Hypoxic depolarization and swelling of the upper blade of the dentate gyrus occur independently from the depolarization in the CA1 region; i.e., spread of the depolarization from CA1 is not required. 4) The hippocampal CA3 region becomes vulnerable to hypoxic depolarization and swelling when excitability is increased sufficiently to trigger episodes of spreading depression before induction of hypoxia or when metabolic activity is severely depressed.
Relative vulnerability of hippocampal formation regions in vitro to hypoxic depolarization
The order of vulnerability to hypoxic depolarization observed in
vitro in the present investigation (CA1 > dentate > CA3) is similar
to that reported in animal models of stroke in vivo (Kirino and
Sano 1984; Pulsinelli et al. 1982
;
Schmidt-Kastner and Freund 1991
). Furthermore, our
results corroborate the longer latencies to hypoxic depolarization
found by others in the dentate gyrus compared with the CA1 region of
the in vitro hippocampal slice preparation (Aitkin and Schiff
1986
; Balestrino et al. 1989
). An important
finding in our experiments was the virtually absolute resistance of
both the CA3 region and the lower blade of the dentate gyrus to
depolarization in response to hypoxia, aglycemia, or the combination of
both insults. Indeed, even 60 min of combined, severe hypoxia and
aglycemia failed to provoke depolarization or swelling in either
region. The differential vulnerability of the upper and lower blades of
the dentate gyrus in vitro has been reported previously by other
investigators (Hara et al. 1990
; Mitani et al.
1994
). Failure of CA3 to swell during hypoxia was also recently
reported by two laboratories (Aitken et al. 1998
; Obeidat and Andrew 1998
). Additionally, the spatial
pattern of both intracellular calcium accumulation and depletion of ATP
during hypoxia in hippocampal slices was identical with the
depolarization and swelling observed in the present experiments
i.e.,
both CA3 and the lower blade of the dentate gyrus were resistant
(Mitani et al. 1994
). It should be noted that earlier
investigations into the susceptibility of individual CA3 neurons to HD
showed conflicting results, which appear to be related to both the
stage of the animal's development and the temperature at which the
slices were preincubated (Davis et al. 1986
;
Janigro and Schwartzkroin 1987
).
Several factors may contribute to the susceptibility of CA1 and the
relative resistance of CA3 to hypoxic-ischemic injury (e.g., see
Crépel et al. 1992). First, neurons in CA3 are
larger and less tightly packed than neurons in CA1, resulting in a
30-50% smaller surface area-volume ratio, a 50% lower neuronal
density, and a 50% greater interstitial volume (Boss et al.
1987
; McBain et al. 1990
;
Pérez-Pinzón et al. 1995
). Thus, even
identical increases in hypoxia-induced ion conductances would produce
smaller changes in Nernst and transmembrane potentials in CA3 neurons when compared with CA1 neurons, thereby leading to reduced
depolarization and associated cell swelling in CA3 neurons. This
concept is further supported by the finding that hypoxic increases in
extracellular [K+] and hypoxic decreases in
extracellular volume are consistently smaller in the CA3 region than in
CA1 (Kawasaki et al. 1990
;
Pérez-Pinzón et al. 1995
). Two additional
factors that may minimize depolarization in CA3 are the relatively low
density of N-methyl-D-aspartate receptors (Cotman et al. 1987
) and the relatively high
density of ATP-sensitive potassium channels in CA3 compared with CA1
(Mourre et al. 1989
). Additionally, the higher level in
CA1 of succinate dehydrogenase, a mitochondrial enzyme involved in
aerobic production of ATP (Kuroiwa et al. 1996
), could
play a role because the postischemic decrease in succinate
dehydrogenase activity is accentuated in CA1, compared with CA3.
Finally, there is more
Na+-K+ ATPase activity in
CA3 compared with CA1 (Haglund et al. 1985
), which
likely improves the ability of CA3 to maintain transmembrane ion
concentration gradients and consequently to resist depolarization.
Association of hypoxic depolarization and optical changes indicative of swelling
HD and rapid decreases in T/T always
corresponded spatially as well as temporally, which had been observed
previously with SD (Snow et al. 1983
). This was
confirmed in two ways: 1) by examining tracings of the
extracellular DC potential and
T/T and
2) by noting the exact position of microelectrodes in images
of the slice relative to the regions undergoing marked changes in
T/T. The consistent coincidence of these
regions suggests that it is reasonable to use the spatial extent of
T/T as an index of the spatial extent of
depolarization. Until recently, the consistent correlation between
changes in light transmittance and swelling strongly implied that cell
swelling was the cause of the changes in
T/T
(Andrew and MacVicar 1994
; Andrew et al.
1996
; Kreisman et al. 1995
; Turner et al.
1995
). However, recent evidence indicates that changes in cell
volume can be dissociated from changes in light transmittance under
certain circumstances in interfaced slices (Aitken et al.
1998
). There is no such dissociation in submerged slices,
wherein the optical responses are reversed in polarity (Obeidat
and Andrew 1998
), probably because the optical properties of
submerged slices are simpler than in interfaced slices. Most important,
the data herein obtained from interfaced slices and those data obtained
from submerged slices (Obeidat and Andrew 1998
) show
that there is a consistent relationship between HD and changes in light
transmittance, regardless of opposite polarities of the optical
response in experiments using different configurations of the
slice-bath interfaces.
Optical changes associated with HD arise from independent foci rather than spread from CA1
Examination of serial images during hypoxia showed that changes in
light transmittance arose independently in the CA1 and upper blade of
the dentate gyrus. The independence of origin was confirmed by imaging
T/T in minislices where CA1, CA3, and the dentate gyrus were physically separated from one another but exposed simultaneously to the same hypoxic stimulus. These experiments show
that spread of depolarization from the CA1 region during hypoxia is not
necessary for induction of depolarization in adjacent structures,
supporting the concept that HD arises independently from multiple foci
(Aitken et al. 1998
). However, the factors that
determine where depolarization is initiated within a given region
remain to be determined.
Prior episodes of spreading depression make the CA3 region vulnerable to HD
In our experiments, the resistance of the CA3 region to HD was
overcome consistently by exposing hippocampal slices to conditions that
triggered episodes of SD, such as severe hypotonia, elevation of
extracellular [K+], or removal of extracellular
Ca2+. Additionally, more severe insults to energy
supply than hypoxia alone, such as pretreating slices with high doses
of DNP, an uncoupler of the mitochondrial electron transport chain,
occasionally fostered the production of HD in CA3. This indicates that
severe depletion of energy supply is a critical factor rendering CA3
vulnerable to HD rather than the occurrence of SDs per se. Confirmation
of this hypothesis necessitates additional experiments that are beyond the scope of this investigation. From the results discussed, three major conclusions can be made about the CA3 region in vitro:
1) the CA3 region in vitro is resistant to depolarization
during severe hypoxia and/or aglycemia. This is supported by a recent histopathological investigation showing that CA3 neurons in vitro are
resistant to hypoxic injury (Newman et al. 1995).
2) Both the capability of the CA3 region in hippocampal
slices to depolarize and the histological appearance of CA3 suggest
that this region is viable. 3) The association between
electrophysiological and histological data suggests that the in vitro
hippocampal slice may be a useful model for investigating early
mechanisms contributing to selective vulnerability and resistance to
hypoxic-ischemic injury.
Our conclusions regarding the CA3 region are of practical significance
because some investigators have reported that neurons in the CA3 region
of hippocampal slices often do not appear histologically as viable as
neighboring CA1 neurons, or CA3 neurons from intact hippocampi fixed in
situ (Newman et al. 1995; Rice et al.
1994
). Significant improvement in the histological appearance
of hippocampal neurons was reported previously after preincubation of
slices in media containing: 1) zero
Ca2+ plus 10 mM Mg2+
(Feig and Lipton 1990
), 2) 3% dextran
(Newman et al. 1995
), or 3) 400 µM
ascorbate (Rice et al. 1994
). In the present
experiments, the CA3 region did not become vulnerable to HD or swelling
after slices were pretreated with media shown by others to improve the histological appearance of CA3 neurons. Additionally, the histological appearance of CA3 neurons in the present experiments was comparable to
that in the treated slices described above. This may be because our
slices were preincubated in the interface position at room temperature
(Newman et al. 1992
; Pohle et al. 1986
).
The inability to record orthodromic population spikes in CA3 in our
experiments also could be interpreted as indicating damage to the
region. However, others have noted that population responses from the
CA3 region are often small in amplitude or absent (Johnston et
al. 1992), whereas intracellular recordings confirm that the pyramidal neurons and their synaptic connections are viable and functioning (Crépel et al. 1992
; Davis et
al. 1986
; Takata and Okada 1995
). The
explanation for this discrepancy is probably related to both the lower
density and nongeometric alignment of CA3 neurons, compared with CA1
neurons
i.e., the varied orientations of individual dipoles
contributing to extracellular field potentials tend to cancel rather
than reinforce each other. Also, the larger extracellular volume
surrounding CA3 neurons (McBain et al. 1990
; Pérez-Pinzón et al. 1995
) tends to reduce
the amplitude of CA3 field potentials. Additionally, more synaptic
contacts to CA3 neurons are probably cut in the preparation of
hippocampal slices because of the wider ramification of their dendritic
trees, compared with CA1 neurons (Johnston et al. 1992
).
Therefore, the absence of orthodromic population spikes cannot be taken
as definitive evidence for the reduced viability of CA3 neurons.
In conclusion, the order of vulnerability of identified regions of the in vitro hippocampal slice to HD and swelling is similar to the pattern of histopathology observed after hypoxia-ischemia in vivo. As such, the results of the present investigation support the use of the hippocampal slice preparation for the investigation of acute changes that may have significance for irreversible hypoxic-ischemic neuronal injury.
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ACKNOWLEDGMENTS |
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We thank Drs. Catherine Cusick, Richard Harlan, and Joseph Weber for help with histology and K. Le for technical assistance.
This investigation was supported by a grant from the American Heart Association, LA Affiliate to N. R. Kreisman and grants from the National Institutes of Health (HD-01072 and HL-62372) and the Maternal and Child Health Bureau (MCJ-229163), and a Career Development Award from the American Lung Association (CI-002-N) to D. Gozal.
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FOOTNOTES |
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Address for reprint requests: N. R. Kreisman, Dept. of Physiology (SL39), Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112-2699.
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 14 July 1999; accepted in final form 21 October 1999.
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REFERENCES |
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