Molecular Brain Research 135 (2005) 285 – 289

www.elsevier.com/locate/molbrainres

Short communication

Valproic acid, but not lamotrigine, suppresses seizure-induced

c-fos and c-Jun mRNA expression
Patricia Szota,b,*, Sylvia S. Whitea, Danny D. Shenc, Gail D. Andersonc
a
Mental Illness Research Education and Clinical Center (MIRECC), VA Puget Sound Health Care System,
1660 South Columbian Way, Seattle, WA 98108, USA
b
Department of Psychiatry and Behavioral Science, University of Washington, Seattle, WA 98195, USA
c
Department of Pharmacy, University of Washington, Seattle, WA 98195, USA

Accepted 18 November 2004

Available online 5 January 2005

Abstract

Seizure-induced activity has been shown to increase the expression of immediate early genes (IEGs) c-fos and c-Jun in the CNS. Anti-
epileptic drugs (AEDs) can suppress the induction of a seizure, but it is unknown if AEDs affect the expression of seizure-induced IEGs. We
found that valproic acid (VPA), but not lamotrigine (LTG), was capable of suppressing seizure-induced c-fos and c-Jun mRNA expression in
rats despite a similar anticonvulsant effect. LTG in some regions of the CNS enhanced seizure-induced IEG expression. These studies
indicate that the older AED (VPA), as compared to the newer AED (LTG), can suppress seizure-induced IEG expression. The consequence of
this suppression of IEGs following a generalized seizure may be viewed either as a neuroprotective or detrimental effect upon the brain.
Published by Elsevier B.V.

Theme: Neurotransmitters, modulators, transporters and receptors

Topic: Signal transduction: gene expression

Keywords: Valproic acid; Lamotrigine; c-fos; c-Jun; Flurothyl; Bipolar disorder; Seizure

Numerous studies have used the expression of the
immediate early genes (IEGs) c-fos and c-Jun as indirect
markers of central nervous system (CNS) neuronal activa-
tion, particularly in response to acute seizure. Following a
generalized clonic–tonic (CT) seizure induced by a con-
vulsant agent such as flurothyl, there is a significant increase
in the mRNA expression of c-fos and c-Jun mRNA in
specific regions of the CNS [17,23,24,28,29]. Anti-epileptic
drug (AED) therapy is commonly used to prevent or
reduce the number of seizures in epileptic patients. How-
ever, if a seizure occurs when AEDs have been admi-
nistered, it is unknown if AEDs will affect the expression of
seizure-induced IEGs. To determine if AEDs can modulate
* Corresponding author. Mental Illness Research Education and
Clinical Center (MIRECC), VA Puget Sound Health Care System, 1660
South Columbian Way, Seattle, WA 98108, USA. Fax: +1 206 768 5456.
E-mail address: szot@u.washington.edu (P. Szot).
0169-328X/$ - see front matter. Published by Elsevier B.V.
doi:10.1016/j.molbrainres.2004.11.011
seizure-induced c-fos and c-Jun mRNA expression, IEG
mRNA expression was measured after flurothyl-induced
generalized CT seizure in rats treated acutely with either
valproic acid (VPA) or lamotrigine (LTG). VPA is one of
the older AEDs used clinically, while LTG is one of the
new generation of AEDs. In addition, both of these agents
are used to treat bipolar disorders [4,21]. VPA is an al-
ternative to lithium in treating acute mania, whereas LTG
is indicated for the treatment of bipolar depression.
Adult male Sprague–Dawley rats (200 g) were purchased
from Bantam and Kingman (Seattle, WA), and housed in
standard cages in a controlled environment with a 12-h
light/dark cycle. Food and water were provided ad libitum.
The animals were given a 7-day acclimating period to the
facility before treatments were started. All animal proce-
dures were in accordance with Animal Care Committee at
the VA Puget Sound Health Care System, Seattle, WA, and
NIH guidelines.
286 P. Szot et al. / Molecular Brain Research 135 (2005) 285–289
Saline (n = 10), valproic acid 250 mg/kg, ip (VPA; n =
10; Sigma, St. Louis, MO), or lamotrigine 10 mg/kg, ip
(LTG; n = 10; generous donation from Glaxo Smith Kline)
were administered by intraperitoneal injection twice a day
for 3 days (approximately 10 h apart). Doses for VPA and
LTG were chosen based on studies examining the effective-
ness of these agents to suppress seizure activity and in
animal studies for bipolar disorder. On the fourth day, 1 h
after the first injection of either saline, VPA, or LTG,
susceptibility to flurothyl-induced seizures was measured as
previously described [24,29] in six of the ten animals from
each group. The latency to the first myoclonic jerk (MJ) and
generalized CT seizure was recorded for each animal in the
three treatment groups. Because flurothyl results in a
progressive increase of seizure activity with exposure, both
of these seizure phenotypes can be observed and latency
recorded for each animal; however, the final seizure that was
observed in animals was a generalized CT seizure. These
animals were sacrificed 1 h after the observance of the
generalized CT seizure, and brains were removed and frozen
on dry ice. Previous work has shown 1 h to be the peak time
for c-fos mRNA expression following a generalized CT
seizure [22,24,28,29]. The remaining four of the ten animals
from each group were not exposed to flurothyl and are
considered the non-seizure controls. These animals were
sacrificed 2 h after injection of either saline, VPA, or LTG to
match the time of sacrifice of the seizure animals. Statistical
comparison of latencies was made using Student’s unpaired
t test with Bonferroni correction for latencies and v 2 test for
seizure latencies.
Brains were cut on a cryostat at 20 Am and alternate
sections were collected onto two sets of slides. Individual
sets of sections were used for c-fos and c-Jun mRNA.
Tissue preparation was conducted as previously described
[28]. The c-fos and c-Jun oligonucleotide probes were 51-
and 45-base sequences, respectively, and complementary to
nucleotides 270–319 for c-fos mRNA [17] or to the last
15 amino acids for the c-Jun mRNA [1]. The oligonucleo-
tide probes were 3V-end-labeled with [33P]-dATP (Perkin-
Elmer, Boston, MA) using terminal deoxyribonucleotidyl
transferase (Invitrogen, Carlsbad, CA) and then purified on
a G25 spin column (Amersham, Arlington Heights, IL). The
c-fos hybridization buffer contained 0.5
106 cpm/50 Al
and the c-Jun hybridization buffer contained 0.65
106
cpm/50 Al. Hyperfilm h-Max (Amersham) was exposed to
slides containing tissue hybridized with either c-fos or c-Jun
[33P] oligonucleotide—24 h for both c-fos and c-Jun. To
quantitate c-fos and c-Jun mRNA expression in specific
regions of the CNS, all sections were processed, hybridized,
and washed in the same session; and each sheet of
Hyperfilm contained sections from the three treatment
groups. Optical densities were measured from films using
MicroComputer Imaging Device (Imaging Research,
Ontario, Canada). Separate optical density measurements
were made of the left and right hemispheres over 3
successive sections, which were anatomically matched
across animals according to the atlas of Paxins and Watson
[20]. Background optical density was subtracted from each
image value for each region. c-Fos mRNA expression was
measured in the cortex (CTX), septum (SEP), caudate
(CAU), hippocampus [CA1, CA2, CA3, and dentate gyrus
(DG)], amygdala (AM), and locus caeruleus (LC). c-Jun
mRNA expression was more limited and analysis was
measured in the DG, AM, and bed nucleus of the stria
terminalis (BNST). Each mean F SEM reported here for
each region in the CNS is the average of six optical density
measurements (after background subtraction) for each
animal. Statistical comparison of c-fos or c-Jun expression
was made using Student’s unpaired t test with Bonferroni
correction for mRNA expression.
Short-term administration of either VPA or LTG signifi-
cantly lengthened the latency to generalized CT seizures
produced by flurothyl as compared to control-treated
animals, although only VPA significantly lengthened the
latency to MJ as compared to control-treated animals
(Fig. 1). Seizure severity as determined by seizure pheno-
type (i.e., progression to tonic extension) or mortality was
not different between treatment groups.
Administration of VPA or LTG alone, with no seizure,
did not affect either c-fos or c-Jun mRNA expression as
compared to saline-treated animals with no seizure. Because
there was no difference in IEG expression in the non-seizure
animals, the optical density values from the three treatment
groups with no seizure were combined and shown in
Figs. 2 and 3 as bno seizureQ. As previously shown [24,29],
a single generalized CT seizure induced by flurothyl in
saline-treated animals significantly elevates c-fos mRNA
expression in many regions of the CNS, including the CTX,
hippocampal CA1, CA3, and DG, AM, SEP, and LC as
compared to non-seizure saline-treated animals.
VPA treatment significantly reduced seizure-induced c-
fos mRNA expression in the CTX, hippocampal CA1,
CA3, and DG, SEP, AM, and LC as compared to saline-
treated animals with a generalized CT seizure (Fig. 2).
The expression of c-fos mRNA in VPA-treated seizure
animals was similar to no seizure animals in all but the
Fig. 1. Flurothyl seizure thresholds in rats treated short term with saline,
VPA, or LTG. Histograms on the left represent the latency to MJ for each
treatment group and the histograms to the right represent the latency to CT
seizure for each treatment group. *Represents statistical ( P b 0.05)
difference to the saline-treated group.
 P. Szot et al. / Molecular Brain Research 135 (2005) 285–289 287

Fig. 2. c-Fos mRNA expression in saline-, VPA-, and LTG-treated rats following flurothyl-induced seizure. (A) Quantitation of c-fos mRNA expression
measured in the cortex (CTX), locus caeruleus (LC), amygdala (AM), septum (SEP), caudate (CAU), and hippocampal CA1, CA3, and dentate gyrus (DG).
Autoradiographic images of c-fos mRNA expression at the level of the septum (B–D) or hippocampus/amygdala (E–G) in saline (B and E)-, VPA (C and F)-, or
LTG (D and G)-treated animals following a seizure. *Represents statistical ( P b 0.05) difference compared to saline-treated animals with a seizure. yRepresents
statistical ( P b 0.05) difference compared to no seizure group.

CTX. Therefore, the administration of VPA suppressed
the generalized CT seizure-induced expression of the IEG
c-fos in most regions of the CNS.
LTG-treated animals with a generalized CT seizure had
c-fos mRNA expression similar to saline-treated animals
with a generalized CT seizure in the CTX, hippocampal
CA1, CA3, and DG, and LC (Fig. 2). However, in the AM,
SEP- and CAU LTG-treated animals with a generalized CT
seizure had significantly more c-fos mRNA expression than
control-treated animals with a generalized CT seizure
(Fig. 2), indicating that LTG either had no effect or
enhanced seizure-induced c-fos expression.
c-Jun mRNA expression following a flurothyl-induced
seizure was limited to fewer regions in the CNS than c-fos
mRNA expression. c-Jun mRNA expression in saline-treated
animals following a seizure was significantly elevated only in
the hippocampal DG and amygdala (AM) as compared to no
seizure animals (Fig. 3). The elevation in c-Jun mRNA
expression in the hippocampal DG induced by a generalized
CT seizure was suppressed by VPA but not LTG (Fig. 3). LTG
significantly elevated seizure-induced c-Jun expression in
the BNST and AM as compared to saline-treated animals with
generalized CT seizure. As with c-fos expression, VPA, but
not LTG, suppressed generalized CT seizure-induced c-Jun
expression, especially in the DG.
These data indicate that VPA was able to prevent the
generalized CT induction of c-fos and c-Jun mRNA
expression in all regions of the CNS studied. For most
regions, seizure-induced IEG expression in VPA-treated
animals (except cortex for c-fos) was identical to animals
with no seizure. In contrast, LTG had no effect on
generalized CT-induced seizure c-fos or c-Jun mRNA
expression in most regions and even increased expression
of these IEGs in other CNS regions.
These results reflect only one dose (versus dose
response) and one convulsant agent. It is possible that this
suppressant response of VPA may not be seen with other
convulsant agents, or that LTG may have a suppressant
effect if other doses were used. A single dose of VPA alone
(no seizure) will increase c-Fos immunohistochemistry in
288 P. Szot et al. / Molecular Brain Research 135 (2005) 285–289

Fig. 3. c-Jun mRNA expression in saline-, VPA-, and LTG-treated rats following flurothyl-induced seizure. (A) Quantitation of c-Jun mRNA expression
measured in the hippocampal dentate gyrus (DG), bed nucleus of the stria terminalis (BNST), and amygdala (AM). Autoradiographic images of c-Jun mRNA
expression at the level of the hippocampus/amygdala (B–D) in saline (B)-, VPA (C)-, or LTG (D)-treated animals following a seizure. *Represents statistical
( P b 0.05) difference compared to saline-treated animals with a seizure. yRepresents statistical ( P b 0.05) difference compared to no seizure group.

the rat hippocampus, but chronic administration has no
effect [3]. This supports the data generated in this study
where 3-day administration of VPA alone had no effect on
IEG mRNA expression. Against noxious thermal stimula-
tion, VPA as well as carbamazepine and phenytoin reduced
the amount of c-Fos immunohistochemistry in the spinal
cord dorsal horn [30]. However, with a generalized CT
seizure, only VPA suppressed IEG expression; but both
AEDs were equally effective at extending the latency to
generalized CT seizure. There is a difference between VPA
and LTG concerning the latency to MJ, but this could not
account for the difference in seizure-induced IEG expres-
sion because the final seizure phenotype that was observed
in the three treatment groups was a generalized CT seizure
which typically increases IEG expression [17,23,24,28,29].
It is unclear why VPA, but not LTG, blocked seizure-
induced IEG expression. Both AEDs affect multiple ion
channels and neurotransmitter systems including the exci-
tatory amino acid, glutamate [7,16,19,21]. Expression of
these IEGs following a seizure has been attributed, in part,
to increased release of glutamate [12,13,25–27,31,33]. If an
anti-glutaminergic effect was the main reason for the
suppression of seizure-induced IEG expression, then this
would suggest that VPA affects the glutaminergic system
differently than LTG. The ability of VPA to suppress
seizure-induced IEG expression may be due to the similarity
of VPA to lithium in treating bipolar disorder [21]; i.e., the
ability attributed to these agents to modulate second
messenger systems such as G-protein-coupled cyclic AMP
and polyphosphoinositol pathways [15,34].
Since c-fos and c-Jun, as transcription factors, are
involved in the pathways for apoptosis and necrosis
[6,8,14,32,35], suppressing expression of these transcription
factors may prevent cell death. Several studies have shown
that blocking the expression of these IEGs results in a
neuroprotective effect [14,32]. Lithium has also been shown
to exert a neuroprotective effect through its ability to
modulate second messenger systems [11,15,18,34]. On the
other hand, it has been shown that VPA and other older AEDs
such as phenytoin and phenobarbital, but not the newer AED
topiramate, result in neurodegeneration in the developing
brain with a similar treatment regimen of twice a day injection
for 3 days [2,5]. In addition, there are data to suggest that a
reduction in IEG expression may be associated with neuro-
degeneration [9,10]. It is possible that the inability of the
brain to respond normally to a stimulus (i.e., increased IEG
expression) may be detrimental, while an AED that allows a
normal response to a stimulus to occur will have less of
disruptive effect upon the CNS. It remains to be determined if
VPA is the only AED that suppresses seizure-induced IEG.
Acknowledgments
This work was supported by the University of Wash-
ington Pediatric Epilepsy Research Center (G.D.A.),
Department of Veterans Affair Puget Sound Health Care
System (P.S.), and National Alliance for Research in
Schizophrenia and Depression (P.S.).
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