Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 4103–4108, April 1997
Genetic determinants of susceptibility to excitotoxic cell death:
Implications for gene targeting approaches
P. E. SCHAUWECKER AND O. STEWARD*
Departments of Neuroscience and Neurosurgery, University of Virginia Health Sciences Center, Charlottesville, VA 22908
Communicated by James L. McGaugh, University of California, Irvine, CA, February 5, 1997 (received for review September 6, 1996)
genes involved in excitotoxic neurodegeneration. Here we
report that certain strains of mice, including strains that are
used for gene targeting studies, do not exhibit excitotoxic cell
death after kainic acid seizures. Kainic acid produced exci-
totoxic cell death in the CA3 and CA1 subfields of the
hippocampus in 129?SvEMS and FVB?N mice, in the same
pattern as described in rats. C57BL?6 and BALB?c mice
exhibited excitotoxic cell death only at very high doses of
kainate, and then only in a very restricted area, although they
exhibited comparable seizures. Hybrids of 129?SvEMS ?
C57BL?6 mice created using embryonic stem cells from
129?SvEMS mice also did not exhibit excitotoxic cell death.
These results demonstrate that C57BL?6 and BALB?c strains
carry gene(s) that convey protection from glutamate-induced
excitotoxicity. This differential susceptibility to excitotoxicity
represents a potential complication for gene targeting studies.
Recent studies have sought to identify the
Excitatory amino acid (EAA) neurotoxicity is thought to play
a key role in secondary degeneration after central nervous
system injury, stroke, and ischemia (1), and also in many
neuropathological disorders, including Alzheimer disease,
Huntington disease, and epilepsy (2). EAA neurotoxicity is
triggered by a massive release of glutamate, which activates
glutamate receptors leading to dramatic increases in intracel-
lular Ca2?(3). The high Ca2?levels initiate signaling cascades
within susceptible neurons that cause neuronal death through
an as-yet-undefined sequence of events (4, 5).
Excitotoxic cell death is most often induced experimentally
by the administration of kainic acid (KA), a potent agonist of
the ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid?
kainate class of glutamate receptors. In rodents, peripheral
injections of KA result in recurrent seizures and the subse-
quent degeneration of select populations of neurons in the
hippocampus (6–8). Thus, KA administration has been widely
used as a model to study EAA neurotoxicity and seizure-
related neurologic diseases (9, 10). More recently, KA neuro-
toxicity has been used as a model to begin to explore the genes
that are involved in EAA neurotoxicity using gene targeting
techniques to create null mutant mice (11–13).
In the course of studies carried out for other reasons, we
have discovered that certain commonly used strains of mice do
not exhibit cell death after KA seizures, whereas others exhibit
neurotoxicity similar to rats. Significantly, the strains involved
are ones that are used for gene targeting studies, raising the
possibility that some of the effects reported in gene targeting
studies (11–13) may, in fact, be due to the genetic background
of the hybrids. The present manuscript characterizes this
MATERIALS AND METHODS
Animals. Male BALB?c and C57BL?6 mice, purchased
from Hilltop Labs (Philadelphia), and male FVB?N and
129?SvEMS mice, purchased from The Jackson Laboratory,
served as subjects. Additionally, mice deficient in the p53
tumor suppressor gene were obtained from The Jackson
Laboratory and hybrid mice (129?SvEMS ? C57BL?6), cre-
ated using embryonic stem cell technology were obtained from
S. Pearson-White (University of Virginia). All mice were
60–90 days old and were housed individually on a 12-h
light?dark schedule. Water and food were available ad libitum.
Drug Administration. KA was dissolved in isotonic saline
(pH 7.3) and administered subcutaneously. Dose–response
studies, in which a range of 20–45 mg?kg of KA was admin-
istered to either FVB?N or C57BL?6 mice, defined seizure
thresholds and mortality rate. Mice were monitored continu-
ously for 4 h for the onset and extent of seizure activity.
Seizures were rated according to a previously defined scale
(14): Stage 1: immobility; stage 2: forelimb and?or tail exten-
sion, rigid posture; stage 3: repetitive movements, head bob-
bing; stage 4: rearing and falling; stage 5: continuous rearing
and falling; stage 6: severe tonic-clonic seizures. These studies
revealed consistent seizures in both strains with a mortality
rate of less than 25% at a dose of 30 mg?kg. All experiments
were performed in accordance with approved institutional
animal care guidelines.
Neuropathological Analysis. At 2, 4, 7, 12, or 20 days
postinjection (n ? 4–10), mice were anesthetized with Nem-
butal (sodium pentobarbital) and perfused transcardially with
4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4).
Horizontal sections were cut on a Vibratome at a thickness of
40 ?m. Every sixth section was stained with cresyl violet to
determine neuronal cell loss, and a representative series of
sections was stained with a modification of the Fink-Heimer
Neuron Counts. Neuron counts were made in areas CA3,
CA1, the dentate hilus, and the dentate gyrus. Only neurons
with a visible nucleus and in which the entire outline of the cell
was apparent were counted. Every sixth section (240 ?m
separation distance) was evaluated with a 63? oil immersion
objective on a Zeiss microscope using a video camera and
placed over the monitor, and the number of neurons contained
within the frame were counted. Cell counts at all levels were
averaged, and mean numbers were used for statistical analysis.
Data are expressed as percent of control (n ? 3–4 control mice
of each strain were evaluated).
Pattern and Extent of Neuronal Activation During KA
Seizures as Revealed by 2-Deoxy[14C]glucose (2DG) Uptake.
Adult male C57BL?6 and FVB?N mice were injected with 5
?Ci of 2DG (New England Nuclear; 323 mCi?mmol) 60 min
per dose) or saline (n ? 4 per strain). Mice were euthanized
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Copyright ? 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA
PNAS is available online at http:??www.pnas.org.
Abbreviations: EAA, excitatory amino acid; KA, kainic acid; 2DG,
*To whom reprint requests should be addressed.
with halothane 45 min after the 2DG administration. The
brains were removed, snap-frozen in isopentane (?15?C) and
stored at ?80?C. Horizontal sections (20 ?m) throughout the
entire rostro-caudal extent of the hippocampus were cut on a
cryostat at ?20?C and were mounted on gelatin-coated cov-
erslips. Every 10th section was thaw-mounted along with14C
standards of known radioactivity on cardboard and exposed to
Kodak Biomax film.
Regional autoradiograph optical density was measured us-
ing the MCID (microcomputer imaging device) image analysis
system (Imaging Research Inc., St. Catherine’s, Ontario, Can-
ada). An average of six measurements per structure were used
on each side, and optical density values were calculated after
subtraction of the film background density. Values given
represent the mean of four mice for each dose and each strain.
Results were assessed statistically by one-way ANOVA, and
intergroup differences were analyzed by Newman–Keuls
Production of Hybrid Mice. 129?SvEMS ? C57BL?6 hybrid
mice were obtained from S. Pearson White. These mice were
generated as part of other gene targeting experiments and
were homozygous wild-type (i.e. control) with respect to the
targeted gene. To produce the hybrids, blastocysts were iso-
lated from C57BL?6 mice on day 3.5 of pregnancy, and 20–25
each blastocoele cavity. The blastocysts were reimplanted into
pseudopregnant 129?SvEMS mice. Chimeric mice were iden-
tified by agouti contribution to the coat color and were
backcrossed to C57BL?6 mice. Germ-line transmission was
determined by the presence of agouti mice in the offspring.
Homozygous null mutants for the p53 tumor suppressor
gene were obtained from The Jackson Laboratory. They were
generated on a 129?Sv background as described previously
(16). The genotypes of the mating pairs had been confirmed
previously using PCR analysis of DNA extracted from mouse
tails (16). Mice homozygous for the targeted p53 mutation
have previously been determined to be null (17–21), and
previous studies have confirmed that these mice do not express
any p53 protein (20, 21).
After systemic administration of 30 mg?kg KA, all four strains
displayed comparable seizures. Within 15 min of the injection,
mice assumed a catatonic posture accompanied by staring
behavior. This behavior was followed by myoclonic twitching
and often frequent rearing and falling. All strains exhibited
continuous tonic-clonic seizures within 1 h of KA administra-
tion, with seizures continuing for 2–3 h. Within 4–5 h after
administration, animals assumed a hunched posture and were
immobile for the next 2–4 h. In all strains, about 75% of the
mice showed generalized clonic seizures within 40–60 min
after administration of 30 mg?kg of KA. Mice not displaying
any type of seizure activity (n ? 1) were not included in the
study, leaving a total of 66 mice that were included in the
analysis at this dose. Table 1 summarizes seizure type and
duration in each strain. Mice did not differ significantly on any
of the measures of seizure severity (F ? 0.541; P ? 0.7118).
To determine whether the LD50of KA is strain dependent,
we administered KA (35, 40, and 45 mg?kg) to C57BL?6 and
FVB?N mice (n ? 10 per group) and assessed seizure severity
and survival rate. In both strains, the LD50 for KA was 35
mg?kg, when administered subcutaneously. At doses greater
than or equal to 35 mg?kg, all mice in both strains (n ? 60)
exhibited class 5 seizures within 10–30 min postinjection. The
duration of seizures increased with the dose of KA adminis-
tered, such that at doses greater than 35 mg?kg, mice exhibited
class 5 seizures for 4–6 h. When 40 mg?kg of KA was
administered less than 30% of both strains survived, and at a
dose of 45 mg?kg, only one mouse in each strain survived. At
both of these doses, mice appeared debilitated 7 days after
administration, and some exhibited spontaneous seizures. At
all doses of KA administered, no significant differences were
found between the duration of seizures, the number of sei-
zures, or the survivability between C57BL?6 and FVB?N mice.
Despite the fact that KA-induced seizures were comparable
across strains, some strains exhibited excitotoxic cell loss while
other strains did not. Both FVB?N and 129?SvEMS inbred
mouse strains exhibited selective excitotoxic cell death com-
parable to what has been described in rats (14, 22, 23). This can
be seen in sections stained to reveal Nissl substance (RNA)
within intact neuronal cell bodies (Fig. 1). For example, the
neuronal cell bodies (Fig. 1 A and C) contained few intact
neurons in KA-treated FVB?N and 129?SvEMS mice (Fig.
1B). Approximately, 50% of the remaining neurons within the
CA3 subfield exhibited signs of degeneration (shrunken nuclei
and pale staining, see Fig. 1D). Cell loss was also evident in the
hilus of the dentate gyrus and in some cases in the CA1 region.
Cell loss was evident throughout the entire rostro-caudal
extent of the hippocampus. In striking contrast, there was no
cell loss or evidence of cellular degeneration in C57BL?6 and
BALB?c mice at doses less than 40 mg?kg (Fig. 1 C and F) at
any time points examined (4–20 days postinjection). These
conclusions were confirmed by quantitative analyses of neuron
numbers (Fig. 2).
Selective silver stains are especially reliable indicators of
excitotoxic cell damage because they stain the debris from
degenerating axons and synaptic terminals and degenerating
neuronal cell bodies. Silver staining of FVB?N and 129?
SvEMS mice revealed degenerating neuronal cell bodies
within the cell layers in CA3 and terminal degeneration within
the neuropil of CA1 and CA3—the principal site of termina-
tion of the projections from neurons in CA3, throughout the
entire rostro-caudal extent of the hippocampus. In about half
of the cases, degeneration debris was also present in the inner
molecular layer of the dentate gyrus—the site of termination
of projections from neurons in the hilus (24) (Fig. 1H).
Degeneration debris was never seen in C57BL?6 or BALB?c
mice at doses less than 40 mg?kg at any of the time points
examined (4–20 days postinjection).
A very small amount of cell death was observed in two of
three C57BL?6 mice that survived after a 40 mg?kg dose of
kainate. A small area of cell loss was observed within area
CA3b (Fig. 3 A and B), but cell loss was restricted to ventral
portions (approximately 200 ?m) of the hippocampus. This
Table 1. Classification of seizure parameters in inbred strains of mice after systemic administration of KA
BALB?c (n ? 11)
C57BL?6 (n ? 24)
FVB?N (n ? 13)
129?SvEMS (n ? 10)
KA induced a similar level of seizure duration and stage irrespective of mouse strain (F ? 0.541; P ? 0.7118).
Seizure parameters, % of mice
4104Neurobiology: Schauwecker and StewardProc. Natl. Acad. Sci. USA 94 (1997)
localized response was confirmed by selective silver staining,
which revealed a discrete patch of degeneration that was
restricted to the ventral portion of the hippocampus (Fig. 3C).
However, the remaining C57BL?6 mouse that survived ad-
ministration of KA at 40 mg?kg did not show any cell loss or
degeneration, and neither did the C57BL?6 mouse that sur-
vived administration of KA at 45 mg?kg.
To ensure that the pattern and extent of neuronal activation
during KA seizures was comparable across strains, we evalu-
ated 2DG uptake after administration of low (5 mg?kg),
intermediate (10–20 mg?kg), and high (30 mg?kg) doses of
KA. At low doses of KA (5 mg?kg), which did not elicit
behavioral seizures, there was no apparent increase in 2DG
uptake within the hippocampus as compared with control mice
(Fig. 4 E and F). In contrast, at higher doses (10, 20, or 30
mg?kg) of KA, increased 2DG uptake was observed through-
although the ventral hippocampus in both strains consistently
showed greater 2DG metabolism than the dorsal hippocam-
pus, similar to what has been described in rats (22). Surpris-
inbred mouse strains, whereas no significant cell loss was observed in any areas examined in BALB?c or C57BL?6 mice (F ? 6.012; P ? 0.005,
cresyl-violet staining of horizontal sections through the hippocampus in a control mouse, (B) in a FVB?N mouse 4 days post-KA injection, and
(C) in a C57BL?6 mouse 4 days after KA administration (30 mg?kg). (D–F) Higher magnification (50?) of the CA3 subfield and the dentate hilar
neurons in an uninjected control mouse, FVB?N, and C57BL?6 mouse, respectively. Note the destruction of neurons in the CA3 subfield and the
dentate hilus (arrows) in FVB?N mice compared with protection against cell loss and degeneration in C57BL?6 mice. (G–I) High magnification
(50?) of the CA3 subfield showing the extent of degeneration in an uninjected control mouse, FVB?N mouse, and C57BL?6 mouse 7 days after
KA administration, respectively. Extensive degenerative debris is present throughout the CA3 subfield in FVB?N mice only. CA1 and CA3 denote
the hippocampal subfields; DG, dentate gyrus. [Scale bars, 750 ?m (A–C); 300 ?m (D–I).]
Neurobiology: Schauwecker and Steward Proc. Natl. Acad. Sci. USA 94 (1997)4105
ingly, a greater metabolic response was observed throughout
doses (10–30 mg?kg; Fig. 5).
Recent studies have used null mutations to identify genes
that may regulate or be associated with the cycle of excitotoxic
cell death. Specifically, it has been reported that a null
mutation of the p53 tumor suppressor gene protects against
neurotoxic cell death after KA administration (12). However,
the null mutant mice were hybrids (129?Sv ? C57BL?6) in
which there is a recombination of 129?Sv and C57BL?6
chromosomes. Because one of the parent strains (C57BL?6)
does not exhibit excitotoxic cell death at doses less than or
equal to the LD50, it is possible that C57BL?6 chromosomes
contain genes other than the null mutation that confer pro-
tection from cell death. To evaluate this possibility, we eval-
uated KA-induced cell death in progeny of 129?SvEMS ?
C57BL?6 created using embryonic stem cells from 129?
genetic background and were the homozygous wild-type mice
(control) with respect to a null mutation created as part of an
unrelated study. After a 35 mg?kg dose of KA, all eight mice
exhibited seizures, but no evidence of degeneration or cell loss
(Fig. 6A), indicating that protection against EAA neurotoxic-
ity is due to the genetic background of the hybrids (specifically,
the presence of C57BL?6 chromosomes).
The protection from excitotoxicity in the p53 null mutant
animals reported in a previous study (12) could have been due
either to the null mutation or the presence of particular
C57BL?6 genes. Thus, we evaluated whether a null mutation
of p53 in a susceptible genotype (129?SvEMS) would convey
protection against cell death. We administered KA to mice
deficient in the p53 tumor suppressor gene that had been
generated in a 129?SvEMS background as described previ-
ously (16–21). As shown in Fig. 6B, silver staining revealed
extensive degeneration (Fig. 6C), and cell loss was apparent in
Nissl-stained sections. After KA administration, extensive
(70–80%) cell loss was observed in the dentate hilus and area
CA3 and was also evident (50% cell loss) in area CA1 (F ?
3.93; P ? 0.001). Thus, null mutations of the p53 gene are not
sufficient to confer protection against KA-induced cell death.
Four commonly used inbred mouse strains demonstrated a
dramatic difference in susceptibility to KA-induced excitotoxic
cell death. C57BL?6 and BALB?c mice were virtually invul-
nerable to KA-induced cell death (except at doses well above
the LD50), whereas FVB?N and 129?SvEMS mice exhibited
excitotoxic cell death similar to what is seen in rats. These
results demonstrate that C57BL?6 and BALB?c strains carry
gene(s) that confer protection from glutamate-mediated ex-
The virtual invulnerability in the two strains cannot be
explained by decreased alterations in the extent of seizure
seizures. Moreover, the pattern and extent of neuronal activity
was comparable as revealed by 2DG autoradiography. Thus,
differences in cell death are not due to differences in blood
brain barrier permeability or differences in the pattern of
neuronal activity during the seizures. The absence of cell death
could, however, be due to differences in EAA receptor func-
tion based on the selective cell loss and degeneration that was
observed in several C57BL?6 mice after injection of KA at
doses exceeding the LD50. It previously has been reported that
at high doses, KA has effects that do not seem to be mediated
via conventional glutamate receptors (25, 26). This might
explain why the pattern of cell loss and degeneration observed
eration is observed in the ventral hippocampus when the dose of
administered KA exceeds the LD50. (A) Low magnification (10?)
cresyl-violet staining of a horizontal section through the hippocampus
in a C57BL?6 mouse 7 days post-KA injection (40 mg?kg). (B) Higher
magnification (66?) of the CA3 subfield showing selective cell loss in
area CA3b (arrows). (C) High magnification (66?) of the CA3
subfield showing discrete degeneration within a subpopulation of cells
in CA3b (arrow). CA1 and CA3 denote the hippocampal subfields;
DG, dentate gyrus; H, hilus. [Scale bars, 600 ?m (A) and 150 ?m (B
In a subset of C57BL?6 mice, select cell loss and degen-
C57BL?6 and FVB?N mice. (A) High magnification (5?) of a
horizontal section showing 2DG uptake in the hippocampus of a
control animal. (B and C) High magnification of a horizontal section
displaying 2DG uptake 60 min after injection of 20 mg?kg KA in a
C57BL?6 and FVB?N mouse, respectively. Metabolism is greatest in
the CA3 and CA1 subfields of the hippocampus, and hippocampal
2DG metabolism is greater in the C57BL?6 mouse (B) as compared
with the FVB?N (C). [Scale bar, 750 ?m.]
Comparison of 2DG response to administration of KA in
4106Neurobiology: Schauwecker and StewardProc. Natl. Acad. Sci. USA 94 (1997)
in the select C57BL?6 mice is not qualitatively similar to what
has been observed in other mouse strains or in rats.
The finding that two commonly used inbred mouse strains
are more resistant to KA-induced excitotoxicity has important
death. It may be that these strains, and any others with similar
resistance, will show very different responses in any situation
in which excitotoxic cell death plays a role (for example, after
brain or spinal cord injury, transient ischemia, or epileptogen-
esis). In this regard, it is of considerable interest that C57BL?6
mice exhibit a substantially different response to spinal cord
injury than rats (27, 28)—a situation in which excitotoxicity is
thought to play an important role (29).
Our results documenting strain differences in susceptibility
to excitotoxic cell death also bear on recent data examining
genes involved in excitotoxicity using null mutations. An
example of this can be seen in the case of null mutations of the
p53 tumor suppressor gene. It has been reported that deletion
of the p53 gene in animals of a mixed genetic background
(129?Sv ? C57BL?6) conferred protection against KA-
induced degeneration (12). Moreover, our findings in hybrids
created with embryonic stem cell technology indicate that
some subset of genes in the 129?SvEMS ? C57BL?6 hybrids
is sufficient to confer protection from excitotoxicity. These
results indicate that the presence of some subset of C57BL?6
genes in the 129?SvEMS ? C57BL?6 hybrids is sufficient to
convey protection from excitotoxicity. It is important to note
that Morrison et al. (12) did report that hybrid animals that
were wild type with respect to the null mutation did exhibit
excitotoxic cell death. The differences in susceptibility to KA
in animals with mixed C57BL?6 and 129?SvEMS genes may be
due to different recombination patterns due to chromosome
sorting in the hybrids. In addition, it is possible that modifying
elements present within the hybrids may affect processes that
involve the null gene. These results substantiate previous
concerns regarding the evaluation of the effects of null mu-
tations in animals with a mixed genetic background (30).
A number of recent gene targeting studies have reported
effects of null mutations on processes that are triggered by or
related to glutamate-mediated excitotoxic cell death. Impor-
tantly, all of these have used animals with mixed genetic
backgrounds in which there may be a contribution of C57BL?6
genes. For example, null mutations of nitric oxide synthase in
129?SvEMS ? C57BL?6 hybrids have been reported to elim-
inate excitotoxic cell death of cortical neurons in culture after
exposure to N-methyl-D-aspartate—an in vitro model of exci-
totoxicity (12). Another study has evaluated the consequences
of a null mutation of c-fos on synaptic reorganization after
seizure-induced excitotoxic death of hippocampal neurons—
again in C57BL?6 ? 129?SvEMS hybrids (14). Although the
mechanism of elicitation of excitotoxicity differs from the
paradigm used in our study, our data suggests that the use of
the hybrids with C57BL?6 genes might influence the resultant
phenotype. These findings will have to be reevaluated in light
genetic background. (A) Silver-stained section from a homozygous
wild-type hybrid mouse (129?SvEMS ? C57BL?6) showing a lack of
degenerative debris after KA administration. (B) Selective silver-
stained horizontal section from a p53?/?mouse generated in a 129?Sv
background illustrating neuronal and terminal degeneration through-
out the CA3 (arrows) and CA1 subfields as well as the dentate hilus
7 days after KA administration. (C) Higher magnification of area CA3
and the dentate hilus (arrows). CA3 denotes the hippocampal subfield.
[Scale bars, 750 ?m (A and B); 200 ?m (C).]
Susceptibility to excitotoxic cell death is dependent on
increased in C57BL?6 mice at intermediate and high doses of KA as compared with FVB?N mice (F ? 50.702; P ? 0.001).
Quantitation of 2DG uptake in the hippocampus in C57BL?6 and FVB?N mice after KA administration. 2DG uptake was significantly
Neurobiology: Schauwecker and StewardProc. Natl. Acad. Sci. USA 94 (1997) 4107
of the present study. To confirm the role that these genes play Download full-text
in excitotoxicity or synaptic reorganization, it will be necessary
to evaluate the consequences of null mutations in mice with a
pure genetic background.
The fact that C57BL?6 and BALB?c inbred strains possess
gene(s) that confer protection against KA-induced excitotox-
icity raises the question of whether similar genes exist in other
species, especially humans. Such genes could account for
differences in susceptibility to neurodegenerative disorders,
seizure disorders, and the response to trauma. It obviously will
be of considerable interest to identify the gene(s) in mice that
are responsible for resistance.
We thank S. Pearson-White for supplying the homozygous wild-type
mice used in this study and H. Scrable for helpful discussions. This
work was supported by National Institutes of Health Grant NS29875
to O.S. and P.E.S. was the recipient of National Research Service
Award Fellowship NS09927.
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