Download Seizure, neurotransmitter release, and gene expression are closely

Epilepsy Research 55 (2003) 117–129
Seizure, neurotransmitter release, and gene expression are closely
related in the striatum of 4-aminopyridine-treated rats
Annamária Kovács a , András Mihály a,∗ , Ágnes Komáromi b , Erika Gyengési c ,
Magdolna Szente c , Roland Weiczner a , Beáta Krisztin-Péva a , Gyula Szabó b ,
Gyula Telegdy b
a
Department of Anatomy, Faculty of Medicine, University of Szeged, P.O. Box 427, H-6701 Szeged, Hungary
b Department of Pathophysiology, University of Szeged, P.O. Box 427, H-6701 Szeged, Hungary
c Department of Comparative Physiology, University of Szeged, P.O. Box 427, H-6701 Szeged, Hungary
Received 22 February 2003; received in revised form 18 June 2003; accepted 22 June 2003
Abstract
The present experiments aimed to compare the length of seizure activity with the time-related increase of transmitter release
and the induction of c-fos gene expression in the striatum of the rat. Anesthetized Wistar rats were intraperitoneally treated with
7 mg/kg 4-aminopyridine, and the transmitter levels in the striatum were measured by means of in vivo microdialysis, 30, 60,
90, 120, and 150 min following the treatment. Striatal and neocortical electric activity was monitored with depth and surface
electrodes, respectively. The expression level of the c-fos gene was estimated by counting the striatal c-fos-immunostained cell
nuclei at the time intervals of the microdialysis. 4-Aminopyridine elicited high-frequency seizure discharges in the EEG and
significantly increased glutamate, aspartate, GABA, serotonin, noradrenaline, and dopamine levels in the extracellular dialysates.
The number of c-fos-stained cell nuclei in the striatum displayed a prolonged increase, showing significantly elevated numbers
throughout the experiment. The increase of c-fos expression in time correlated best with the increase of glutamate release,
which was also significantly elevated at every sampling time. The GABA release, culminating at 60 min after the seizure onset,
correlated best with the cessation of the electrographic seizure. Aspartate, norepinephrine, serotonin, and dopamine displayed
transient but significant elevations. We conclude that glutamate plays the essential role (most probably through ionotropic and
metabotropic receptors) in the extracellular signaling, which eventually leads to intracellular cascades and c-fos gene expression
in the striatum during convulsions.
© 2003 Elsevier B.V. All rights reserved.
Keywords: 4-Aminopyridine; Glutamate; Seizure; c-fos; Seizure-related glutamate release
Abbreviations: 4-AP, 4-aminopyridine; ARG, arginine; ASP, asparatic acid; CIT, citrulline; DA, dopamine; DOPA, 3,4-dihydroxyphenylalanine; DOPAC, 3,4-dihydroxyphenylacetaldehyde; GABA, 4-aminobutyric acid; GLU, glutamic acid; 5-HIAA, 5-hydroxyindole-3-acetate;
5-HT, serotonin (5-hydroxytryptamine); HVA, homovanillic acid; ITF, inducible transcription factor; 3-MT, 3-methoxytyramine; NE,
norepinephrine; NMDA, N-methyl-d-aspartate; NO, nitric oxide; NOS, nitric oxide synthase
∗ Corresponding author. Tel.: +36-62-545-670; fax: +36-62-545-707.
E-mail address: [email protected] (A. Mihály).
0920-1211/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0920-1211(03)00113-X
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A. Kovács et al. / Epilepsy Research 55 (2003) 117–129
1. Introduction
Excitatory amino acids (EAAs) such as glutamic
acid and aspartic acid take part in the induction,
maintenance, and propagation of seizures and mediate some of the neuropathological effects of neuronal
hyperactivity (Sloviter and Dempster, 1985; Peña and
Tapia, 2000). Glutamate (GLU) and aspartate (ASP)
are epileptogenic and excitotoxic to nerve cells both
in vitro and in vivo (Whetsell, 1996). The release
of these EAAs from the nerve terminals may be
Ca2+ -dependent or -independent, although the mechanisms are not fully understood. Extracellular K+
stimulates the EAA efflux from striatal neurons (Korf
and Venema, 1985), but the GLU release may depend
on many other molecular signals, such as the release
of nitric oxide (NO; McNaught and Brown, 1998), the
activation of presynaptic metabotropic GLU receptors
(Cartmell and Schoepp, 2000), 4-aminobutyric acid
(GABA) receptors and protein kinases (Perkinton and
Sihra, 1999).
It was believed for years that the main pathogenetic
factor in epilepsies was the impairment of GABAergic transmission (Meldrum, 1975). It turned out later
that inhibitory neurotransmission may play an important role in the generation and maintenance of certain
seizure types in the hippocampus and in the corticothalamo-cortical circuits (Avoli, 1996; Engel, 1996).
Recently, more fundamental processes underlying
epileptic phenomena came into focus: the role of
neuronal ion channels, and particularly K+ channels,
which regulate neuronal excitability and are coupled
to several transmitter systems (Hoffmann et al., 1997).
Immunohistochemical studies have proved the presence of these channels on the processes of the neuron
(Betancourt and Colom, 2000) and we are now only
beginning to understand the significance of their local
distribution and density on the surface of the neuron.
4-Aminopyridine (4-AP) is a convulsant which
blocks some of the voltage-dependent neuronal potassium channels: the KA channel (or A channel), which
regulates the spike frequency in postsynaptic structures; the KV channel (or delayed rectifier), involved
in the repolarization phase of the action potential;
and the KACh receptor-coupled channel (Alexander
and Peters, 2000). The effects of 4-AP, therefore, include long presynaptic depolarization and increased
transmitter release (Thesleff, 1980). It has been proved
experimentally that 4-AP stimulates the release of
GLU (Tibbs et al., 1989; Patterson et al., 1995), norepinephrine (NE; Versteeg et al., 1995), serotonin
(5-HT; Schechter, 1997), and dopamine (DA; Bonnano
et al., 2000). In the hippocampus, 4-AP increases the
release of GLU, ASP, and GABA in a dose-dependent
manner, and causes typical convulsive EEG changes
(Peña and Tapia, 1999, 2000). In contrast, 4-AP did
not increase the extracellular concentration of GLU
in the striatum of freely moving rats (Segovia et al.,
1997).
The experimental convulsion evoked by 4-AP is followed by the appearance of the inducible transcription factor (ITF) c-fos in neuronal cell nuclei (Mihály
et al., 1997, 2001; Szakács et al., 2003), and by an
increased regional cerebral blood flow in those brain
areas which participate in the spread of the seizures
(Mihály et al., 2000). However, there is no direct in
vivo evidence of a causal relationship between neurotransmitter release and the ITF expression induced by
4-AP. Accordingly, the present experiments were designed to measure the extracellular concentrations of
neurotransmitters and their metabolites via an in vivo
microdialysis technique and to detect the expression of
c-fos by means of immunohistochemistry in the same
brain area under the same experimental conditions.
The striatum was chosen because it plays an important
role in the motor coordination and has abundant cortical and subcortical connections which are strongly
activated in motor seizures (Kusske, 1979; Hosokawa
et al., 1983; Bonhaus et al., 1986).
2. Materials and methods
Adult male Wistar rats weighing 200–250 g were
housed in a light- and temperature-controlled room
(lights on between 6:00 a.m. and 6:00 p.m.; 23 ◦ C) and
had free access to food and water. The animals were
kept and handled during the experiments in accordance
with the standards of the Ethical Committee for the
Protection of Animals in Research in Szeged University. The intraperitoneal (i.p.) injection of awake
animals with 7 mg/kg 4-AP produced intense motor alterations (tremor, shivering and generalized
tonic–clonic seizure; Mihály et al., 1990), and therefore the electrophysiological, microdialysis and immunohistochemical experiments were carried out in
A. Kovács et al. / Epilepsy Research 55 (2003) 117–129
separate groups of rats anesthetized with Nembutal
(35 mg/kg), injected i.p. The body temperature was
maintained at 37 ◦ C by using a heating pad. The
anesthesia was maintained with repeated Nembutal
(15 mg/kg) injections throughout the experiment. The
experiments were performed in separate groups of
animals, as listed below.
2.1. Electrophysiology
Four animals were used for a direct test of the convulsive effect of 4-AP. The rats were anesthetized with
Nembutal (35 mg/kg) and a steel needle depth recording electrode was inserted into the left striatum (interaural 9.48 mm, bregma 0.48 mm, depth 5.50 mm;
Paxinos and Watson, 1998) with the help of a stereotaxic apparatus. The EEG electrodes were placed on
the surface of the skull (see Fig. 2). The 7 mg/kg 4-AP
was injected intraperitoneally to the anesthetized rats
and recordings were made at 15-min intervals, with
10-min interruptions, for 150 min. The position of the
depth electrode was checked after the experiment, similarly to that of the microdialysis probe.
2.2. Microdialysis
Fourteen animals were used for microdialysis: seven
were treated with 7 mg/kg 4-AP, while seven control
animals were injected with physiological saline. The
microdialysis probe was implanted into the right striatum (interaural 9.48 mm, bregma 0.48 mm; Paxinos
and Watson, 1998) under Nembutal (35 mg/kg) anesthesia. The probe was located in the caudate–putamen
region, and never penetrated the accumbens region
or the olfactory tubercle (see Fig. 5). The probe was
continuously perfused with modified Ringer solution
(composition: 140 mM NaCl, 3.0 mM KCl, 1.2 mM
Na2 HPO4 , 1.0 mM MgCl2 , 7.2 mM glucose, 1.2 mM
CaCl2 , pH = 6.8–7.2) at a flow rate of 2 ␮l/min,
delivered by a CMA 100 microinjection pump. After
a 2-h equilibrium period, perfusates were collected
in polyethylene vials every 30 min throughout the
experiments. Three baseline samples were obtained
from every animal before the administration of 4-AP
(7 mg/kg, i.p.). The average of these samples were
depicted as 100% during the measurements, and the
seizure-induced changes were expressed as percentages and handled as such in every animal, because
119
of the inter-animal variances. Immediately following
sampling, dialysates were assayed for levels of GLU,
ASP, arginine (ARG), citrulline (CIT), and GABA via
high-performance liquid chromatography with electrochemical detection (HPLC-EC) following precolumn
o-phthalaldehyde-sulfite derivatization at room temperature for 15 min. Amino acids were separated by
using a reverse-phase column (250 mm×4 mm, Nucleosil 5C18) and a mobile phase consisting of 100 mM
monosodium phosphate and 0.5 mM EDTA with 25%
methanol (v/v) in water adjusted to pH 4.95 with 85%
phosphoric acid. The total elution time was 20 min.
The column temperature was kept at 25 ◦ C. Brain
dialysate samples were also assayed for levels of DA,
NE, 5-HT, 3,4-dihydroxyphenylalanine (DOPA), 3,4dihydroxyphenylacetaldehyde (DOPAC), homovanillic acid (HVA), 5-hydroxyindole-3-acetate (5-HIAA),
and 3-methoxytyramine (3-MT) by HPLC-EC. Samples were separated by using a BST (60 mm × 4 mm)
Hypersil 3ODS reverse-phase analytical column
and a BST (20 mm × 4 mm) Hypersil 3ODS guard
column and a mobile phase consisting of 100 mM
Na2 HPO4 , 0.7 mM EDTA, 0.7 mM octane sulfonic
acid with 12% methanol (v/v) in water adjusted to pH
2.90 ± 0.05 with 85% phosphoric acid. The column
temperature was 25 ◦ C. The total elution time was
15 min. The results are expressed as means ± S.E.M.
of the basal levels as percentage. Statistical significance was determined by one-way ANOVA, using
Tukey’s Studentized Range (HDS) test. A P value of
less than 0.05 was regarded as significant.
At the end of the experiments, the rats were perfused transcardially with 4% paraformaldehyde in
0.1 M phosphate buffer, pH 7.2. The brains were
postfixed overnight, and 20 ␮m thin coronal sections
were cut on a Reichert Jung freezing microtome.
The site of the implanted probe was investigated on
Nissl-stained frozen sections. Some of these frozen
sections were stained with c-fos immunohistochemistry in order to reveal the effect of the tissue damage
caused by the probe.
2.3. c-fos immunohistochemistry
In separate experiments, 15 male rats (200–220 g)
were anesthetized with Nembutal, and injected
i.p. with 7 mg/kg 4-AP in physiological saline. Controls (another 15 animals) received the solvent only.
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The animals were subjected to transcardial perfusion
with 4% paraformaldehyde in 0.1 M phosphate buffer
(pH 7.4) 30, 60, 90, 120, and 150 min after the treatment: three controls (saline-treated) and three convulsing (4-AP treated) animals were perfused at every
time. The Nembutal anesthesia was maintained with
repeated i.p. injections throughout the experiment.
The brains were dissected and postfixed for 1 h at
room temperature. Following postfixation, the brains
were cryoprotected overnight in 30% sucrose in 0.1 M
phosphate buffer. Frozen serial sections at a thickness
of 24 ␮m were cut on a cryostat in the coronal plane,
and one of two sections was processed for immunohistochemistry according to the following protocol.
Single primary antibody (polyclonal c-fos antibody
raised in rabbit, 1:1000; Santa Cruz Biotechnology,
CA) was followed by secondary antibody (donkey
anti-rabbit IgG, 1:40; Jackson ImmunoResearch,
PA), with detection by the peroxidase-anti-peroxidase
(PAP; Jackson ImmunoResearch) method (PAP dilution 1:1000), and the peroxidase reaction was
developed by using nickel-intensified 3 3 -diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO)
as chromogen.
2.4. Analysis of the immunostained sections
The brains of three animals in each experimental group (30, 60, 90, 120, and 150 min following
4-AP and saline injections; 30 animals) were investigated. Sections of identical coronal stereotaxic planes
(Paxinos and Watson, 1998) were used. Counting in
the striatum was performed blindly of the animal’s
treatment, on three histological sections from every animal (i.e., nine tissue samples from each time
group). Rectangular areas (the image capturing field
of the camera) were selected from the whole striatum
(Fig. 1), and the immunoreactive nuclei displaying grayish-black staining were counted by using a
NIKON ECLIPSE 600 microscope equipped with a
Polaroid DMC digital camera (1600 dpi × 1200 dpi
in 8 bits) with 20× objective magnification, with the
Image Pro Plus 4 morphometry program (Media Cybernetics, Silver Spring, MD). Following background
subtraction, the threshold was adjusted so that paleand deep-stained nuclei could be recognized equally
by the counting program. The rectangular image
capturing fields were equal to 0.249 mm2 with the
magnification used. The cell counts were analyzed by
Fig. 1. Sampling method for the counting of the c-fosIR cell nuclei. The rectangles in the striatum represent the image-capturing field of
the camera (AOI). This AOI size is used for counting with 20× objective magnification. As shown by the drawing, 13 AOI’s are selected
from every section, which cover the whole caudate–putamen region (with the exception of the ventral striatum and pallidum, which were
not investigated).
A. Kovács et al. / Epilepsy Research 55 (2003) 117–129
means of ANOVA (post hoc test: Bonferroni method).
The statistical analysis was performed with the help
of the SPSS 9.0 computer program.
3. Results
3.1. Electroencephalography
The depth and EEG recordings proved that the
ictal activity in the neocortex and striatum began
15–25 min following the 4-AP injection (average latency 18 min). The background activity fell markedly
and high-frequency tonic–clonic activity developed
(Fig. 2). The amplitude of the ictal discharges increased and reached a maximum of 1.5–2.0 mV. The
striatal seizure began 2–4 s later than the neocortical
one (Fig. 2). The electric seizure phenomena greatly
diminished after 50 min (from the beginning) and
were completely absent from 60 min up to the end
of the experimental period (Fig. 2). However, in the
striatum spiking activity was continuous throughout
the experiment: the amplitude of the spikes was less
than 1 mV, but their frequency was 3–4 Hz even at the
end of the experiment (Fig. 2B).
121
3.2. Microdialysis: transmitter amino acids
The GLU concentration of the dialysate was significantly elevated at every sampling time following 4-AP
injection. Maximum levels were measured at 120 min.
The GLU levels in the control animals were constant
(Fig. 3). Measurements of ASP resulted in a different curve: a sharp early rise in ASP concentration at
30 min which returned to the control level, and a later
small, but significant elevation at 90 min (Fig. 3). The
concentration of GABA displayed a steady increase
and reached a maximum at 60 min. This 60-min value
was significantly higher than that of the control. After 60 min, it decreased, and remained at an elevated
level, which did not differ significantly from the control (Fig. 3).
3.3. Nitric oxide-related amino acids
Following the 4-AP injection, the concentration of
CIT peaked at 90 min with a significant elevation,
then quickly returned to the control level at 120 min
(Fig. 3). The level of ARG exhibited a slow elevation,
which became significant at 60 min, and remained elevated until the end of the experiment (Fig. 3).
Fig. 2. Seizure activity evoked by 4-AP in the striatum and neocortex. (A) The ictal activity begins 17 min after the 4-AP injection. (B)
Disappearance of the epileptiform activity by the end of the experiment. Note that frequent, low amplitude spikes are detected with the
striatal electrode only. (C) Schematic view of the arrangement of the recording electrodes (ch1: needle electrode inserted into the striatum;
ch2, 3, 4: surface electrodes above the neocortex; calibration applies to panels A and B).
A. Kovács et al. / Epilepsy Research 55 (2003) 117–129
300
GLU
*
*
*
**
*
200
base
base %
250
%
122
150
100
50
0
30
60
90
120
350
300
250
200
150
100
50
0
*
150
0
30
time (min)
control (5)
GABA
4-AP (6)
control (5)
300
**
200
90
120
150
4-AP (4)
CIT
250
200
base
base %
250
60
time (min)
%
300
ASP
*
150
150
100
100
50
50
0
30
60
90
120
0
150
30
control (4)
control (5)
4-AP (6)
base %
250
60
90
120
150
time (min)
time (min)
4-AP (4)
ARG
200
**
*
150
*
100
50
0
30
60
90
120
150
time (min)
control (5)
4-AP (6)
Fig. 3. Changes in concentrations of glutamate (GLU), aspartate (ASP), 4-aminobutyric acid (GABA), l-citrulline (CIT), and l-arginine
(ARG) in control and 4-AP-treated rat striatum, expressed as percentages of the basal level. The number of dialysis samples (4–6) is given
in brackets, and the injection of 4-AP or physiological saline is indicated by arrows. Asterisks denote significant differences: ∗ P < 0.05;
∗∗ P < 0.01.
3.4. Catecholamines, serotonin, and metabolites
In the rat striatum, 4-AP treatment resulted in an increased release of all of the catecholamines and their
metabolites, except HVA. The concentrations of DA,
DOPA, and 3-MT remained increased throughout the
whole test period, but only the DOPA concentration increase remained significant until the end of the experiment. DA release was initially highly significant, by
150 min it had decreased but remained elevated compared to the control (Fig. 4). The amounts of striatal
DOPAC and 5-HIAA revealed steady increases and at
the end of the experiment significant increases were
seen (Fig. 4). The extracellular level of NE rose significantly immediately after 4-AP administration, but
had fallen to the control level at 60 min and did not
change thereafter (Fig. 4). The increase in the 5-HT
level was similar to that in DA, showing a long-lasting
significant elevation, but its decrease to the control
level occurred earlier (Fig. 4).
3.5. c-fos immunoreactivity in the striatum
The implantation of the probe elicited tissue damage, and scattered c-fos immunoreactive (c-fosIR) cells
were visible around the tissue damage (Fig. 5). These
350
300
250
200
150
100
50
DOPA
*
*
*
DA
***
30
60
200
*
100
30
60
90
120
0
150
control (5)
control (4)
4-AP (7)
DOPAC
*
*
120
150
base %
300
250
200
150
100
50
0
30
60
90
140
130
120
110
100
90
80
control (4)
0
150
4-AP (6)
30
60
90
120
150
time (min)
control (4)
4-AP (5)
500
3-MT
*
400
base %
*
120
HVA
time (min)
300
90
time (min)
time (min)
base %
*
0
0
base %
123
300
*
base %
base %
A. Kovács et al. / Epilepsy Research 55 (2003) 117–129
200
100
4-AP (5)
NE
300
200
100
0
0
0
30
60
90
120
0
150
4-AP (6)
control (4)
300
5-HT
**
200
100
0
0
30
60
90
120
90
120
150
150
200
180
160
140
120
100
80
4-AP (5)
*
5-HIAA
*
0
30
60
90
120
150
time (min)
time (min)
control (4)
60
control (5)
base %
base %
**
30
time (min)
time (min)
4-AP (5)
control (4)
4-AP (6)
Fig. 4. Changes in concentrations of 3,4-dihydroxyphenylalanine (DOPA), dopamine (DA), 3,4-dihydroxyphenylacetaldehyde (DOPAC),
homovanillic acid (HVA), 3-methoxytyramine (3-MT), norepinephrine (NE), 5-hydroxytryptamine (5-HT), and 5-hydroxyindole-3-acetate
(5-HIAA) in control and 4-AP-treated rat striatum, expressed as percentages of the basal level. The number of dialysis samples (4–7)
is given in brackets, and the injection of 4-AP or physiological saline is indicated by arrows. Asterisks denote significant differences:
∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
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A. Kovács et al. / Epilepsy Research 55 (2003) 117–129
Fig. 5. Immunohistochemical detection of c-fos in the striatum. (A) An animal perfused after the 4-AP plus microdialysis experiment
displays the track of the microdialysis probe (arrow) and scattered c-fos staining around the probe. Note the strong c-fos immunostaining of
the neocortex (cx) and piriform cortex (Pi). LV: lateral ventricle. Bar: 1 mm. (B) An animal perfused 90 min after 4-AP injection without
microdialysis. The striatum contains strong c-fos immunoreactivity, represented by immunostained cell nuclei. Bar: 200 ␮m.
c-fos IR cell number / mm
2
animals were not used for quantitative immunohistochemistry because the tissue damage itself elicited
c-fos expression and disturbed the counting.
Control animals (not injected with 4-AP) displayed
very few c-fosIR cell nuclei scattered in the striatum.
500
450
400
350
300
The animals which received 4-AP treatment under
Nembutal anesthesia exhibited a characteristic c-fos
expression pattern. The number of c-fosIR cells gradually increased from 30 min, and reached a maximum
at 60 min (Fig. 6). A decrease was observed thereafter,
*
*
*
4-AP
*
250
200
150
100
50
0
control
*
*
*
*
*
30
60
90
time (min)
120
150
Fig. 6. The number of c-fos immunoreactive (c-fosIR) cell nuclei in the striatum of anesthetized rats following 4-AP injection without
microdialysis. Controls were injected with physiological saline. Every value differs significantly (P < 0.01) from the others. The S.E.M.
values are shown at the top of the columns. The number of c-fos immunoreactive cells is largest at 60 min, then displays a gradual decrease,
but the 150-min value is still significantly higher than the control.
A. Kovács et al. / Epilepsy Research 55 (2003) 117–129
but at the end of the experiment (at 150 min) the level
was still significantly higher than the control (Fig. 6).
We did not attempt to characterize the cells expressing
c-fos protein. The size of the nuclei suggested that all
of them were neuronal cell nuclei. The seizure-induced
elevations in the number of c-fosIR cells were significant as compared not only to the controls, but also
to each other at every time interval (Fig. 6). In the
present experiments, no efforts were made for the
co-localization of c-fosIR with neuronal antigens, or
for the investigation of the relation of c-fosIR to the
matrix and striosome compartments.
4. Discussion
The effects of experimental seizures on transmitter
release have been investigated by a number of authors
(Nitsch et al., 1983; Jacobsson et al., 1997; Segovia
et al., 1997; Smolders et al., 1997; Morales-Villagrán
et al., 1999; Peña and Tapia, 2000). It is clear that
acute experimental convulsions increase the extracellular concentrations of various transmitter substances
because of the prolonged neuronal depolarization and
the elevated extracellular K+ concentration (Macı́as
et al., 2001). Nuclear magnetic resonance studies
have revealed that seizures in the human neocortex
increase the concentrations of transmitter amino acids
(Sherwin, 1999). Our experimental data confirm these
results and supply further evidence for the complex
metabolic action of the seizure. The results also emphasize the importance and compatibility of animal
experiments with regard to the human disease.
4.1. Electrophysiology
The present data prove that i.p. injection of 4-AP
precipitates electrographic seizure in the striatum and
neocortex. The beginning of the striatal seizure can be
detected 2–4 s after the onset of the neocortical ictal
phenomena, indicating the spread of seizure activity
from the neocortex towards the striatum. The excitatory glutamatergic afferents of the striatum originate
from the neocortex and thalamus (Flores-Hernández
et al., 1994) explaining that the neocortical and striatal
EEG seizure events showed synchrony and both disappeared 50–60 min after the 4-AP injection. However,
neuronal spiking activity persisted in the striatum with
125
higher frequencies than in the neocortex, indicating
that the continuous in vivo release of several transmitters and metabolites caused a long-lasting excitation
of striatal projection neurons (Flores-Hernández et al.,
1994).
4.2. Glutamate and aspartate
GLU and ASP are natural ligands and agonists of
the ionotropic and metabotropic glutamate receptors
(Alexander and Peters, 2000). GLU plays an important role in seizure initiation and spread exerting action
not only on ionotropic, but also on metabotropic receptors (Chapman, 1998). The rise of GLU (and ASP)
concentration in chronic seizures is well established
(for a review, see Chapman, 1998). However, GLU is
not always elevated in acute seizures (Nitsch et al.,
1983; Segovia et al., 1997). Our present experiments
demonstrate fast and highly significant elevation, indicating the transmitter role of GLU in the striatum.
Since many of the excitatory afferents of the striatum
originate from the cerebral cortex (and thalamus), we
may suppose that the elevation in the striatal GLU is a
consequence of neocortical hyperactivity and the excessive release of the transmitter from corticostriatal
(and thalamostriatal) axon terminals. Interestingly, the
GLU level reaches its maximum at 120 min, well after
the cessation of the electrographic seizure. This explains the long-lasting spiking activity on the striatal
EEG and raises the possibility that besides the postsynaptic depolarization, GLU might regulate the release
of other transmitters through metabotropic GLU receptors (Pisani et al., 2002). Recent experimental evidence proves that protein kinase C (the activation of
which is coupled to metabotropic GLU receptors) is
an important mediator of the phosphorylation of transcription factors which are responsible for c-fos gene
expression (Choe and Wang, 2002); so, metabotropic
receptors themselves (subtypes 1 and 5; Cartmell and
Schoepp, 2000) can participate in the induction of
c-fos expression.
A significant elevation was detected in the ASP
level too, although the dynamics of ASP release displayed a different pattern: an early rise and fall, and
then a much smaller (but significant) elevation. This
observation is in register with literature data on the
increase of ASP release in the striatum following
electrical stimulation of the neocortex (Perschak and
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Cuénod, 1990). ASP, if released from nerve terminals acts mainly on NMDA-type receptors, so it may
contribute to neuronal c-fos expression in seizure
(Gundersen et al., 2001). On the other hand, ASP
serves as important intermediate in energy metabolism
of the brain—it is produced mainly from oxaloacetate
by the enzyme aspartate aminotransferase, and its
production is enhanced in hypoglycemia; the number
of ASP immunoreactive neurons increases significantly in the hypoglycemic striatum (Gundersen et al.,
2001).
4.3. GABA
Pharmacological data suggest that in some acute
epilepsy models the GABA levels decrease (Rowley
et al., 1995), whereas in other experiments increased
GABA levels were observed (Smolders et al., 1997).
Recent studies indicate that GABA has an important
role in epileptogenesis: GABA-mediated neuronal
synchronization initiates seizures in some forebrain
areas, mainly through the activation of GABAA receptors (Avoli, 1996). In the present experiments,
the GABA levels rose slowly and peaked at 60 min,
which indicated that GABA was released from striatal
interneurons (Smith and Bolam, 1990) upon the effect
of afferent excitatory axon terminals (Heimer et al.,
1995). Although there are no literature data concerning the ratio of the GLU- and GABA-containing
axon terminals, one explanation of our present observation may be that the number of GLU terminals
is far greater than that of the GABAergic terminals;
the detectable amount of GABA is, therefore, smaller
and an increase to detectable levels takes more time.
Interestingly, the GABA peak coincides with the
cessation of the electrographic seizure (60 min), indicating the importance of GABAergic neurons in the
control of and (probably) protection against striatal
seizures.
4.4. Nitric oxide-related amino acids
NO regulates many physiological processes and also
contributes to the neurotoxicity of GLU in pathological conditions (Bredt and Snyder, 1992). NO is synthesized in the brain by isoforms of NO synthase (NOS)
which converts ARG to CIT (Bredt and Snyder, 1992).
In the striatum, some of the large, aspiny interneurons
contain neuronal NOS (Vincent and Johansson, 1983).
We observed a significant, steady, slow increase in
ARG, and a sharp CIT peak at 90 min. This probably
indicates the slow activation of NOS and the elevation
of intrastriatal NO levels. Seizure conditions elevate
brain NO levels (Kashihara et al., 1998), and the inhibition of NOS decreases seizure-related GLU release
(Rigaud-Monnet et al., 1995). In the present experiments, GLU release increased first (at 30 min), the
level of ARG increased 30 min later (at 60 min), then
a CIT peak occurred only subsequently (at 90 min).
This timing suggests that GLU release activated NOS,
which led to the enhanced conversion of ARG to
CIT. This result correlates well with the literature data
on the NMDA sensitivity of NOS-containing neurons
(Kendrick et al., 1996).
4.5. Catecholamines and metabolites
It is known from the literature that 4-AP increased the spontaneous release of DA in rat brain
slices, which could be a consequence of the depolarizing action of 4-AP on dopaminergic axon
terminals (Versteeg et al., 1995). Accordingly, the
precursor DOPA and the metabolites DOPAC, 3-MT,
and HVA also underwent a slow accumulation, although the HVA levels did not increase significantly;
proving the activity-related upregulation of DA synthesis and degradation by monoamino-oxidase and
catechol-o-methyltransferase. The DA terminals originate in the substantia nigra, which is activated strongly
in seizures (Bonhaus et al., 1986). This activation
probably also contributes to the prolonged increase of
striatal DA level. DA (probably through D4 receptors)
prevents hyperexcitability (Rubinstein et al., 2001)
and has a clear-cut neuroprotective role (Bozzi et al.,
2000). On the other hand, we have to consider that
whilst DA decreases striatal convulsion, it may also
suppress the induction of c-fos in striatal neurons,
probably through D2 receptors (Adams and Keefe,
2001; Marshall et al., 2001). Thus, the prolonged DA
release probably contributed not only to the cessation
of the electrographic seizure, but also to the fact, that
the number of c-fosIR neurons was medium in the
striatum (as compared to the c-fosIR neuron numbers
in the cerebral cortex; Szakács et al., 2003).
4-AP increases NE release both in vitro (Versteeg
et al., 1995) and in vivo (Morales-Villagrán et al.,
A. Kovács et al. / Epilepsy Research 55 (2003) 117–129
1999). The time course of NE release in neocortical seizures (Morales-Villagrán et al., 1999) is very
similar to that we measured in the present experiments. Striatal NE innervation originates from the locus ceruleus (Heimer et al., 1995), and NE is known
to exert anticonvulsant activity, probably through beta
receptors (Ferraro et al., 1994). However, this anticonvulsant effect did not last longer than 40–60 min
in our experiments, because the increased NE in the
dialysates had returned to the base level at 60 min.
Nevertheless, we think that DA and NE together are
able to exert anticonvulsant and neuroprotective roles,
and probably contribute to the cessation of the electrographic seizure and the decrease in c-fos immunoreactivity during the experiments.
4.6. Serotonin and metabolites
Serotoninergic afferents to the striatum originate in
the dorsal raphe (Heimer et al., 1995). The axons display a high degree of collateralization (Heimer et al.,
1995). Similarly to that of DA, 5-HT release is enhanced by 4-AP (Versteeg et al., 1995). Our present
results prove that the characteristic features of 5-HT
release are similar to those of DA and GLU, indicating that 4-AP acts on axon terminals in the striatum. 5-HIAA, a metabolite of 5-HT, was present in
increased amount in the dialysates, indicating the extracellular degradation of the transmitter. 5-HT plays
an important role in the striatum, regulating the release
of GLU (Di Cara et al., 2001) and DA (Sershen et al.,
2000). Seizure experiments demonstrate the modulatory role of 5-HT through different receptors: its inhibitory effect is mediated through 1A receptors to
open Ca2+ -independent potassium channels (Colino
and Haliwell, 1987). The same receptor can reduce
the presynaptic Ca2+ entry into glutamatergic terminals, and thereby inhibit the release of GLU (Schmitz
et al., 1997).
127
activity (Labiner et al., 1993; Mihály et al., 1997,
2001). Our experiments indicated that the release of
GLU showed the best correlation with the increase
of c-fosIR cells in the striatum. Pharmacological experiments in our laboratory proved the important role
of ionotropic GLU receptors in the expression of the
neuronal c-fos gene (Szakács et al., 2003). Therefore,
we think that the gradual increase of the number
of c-fosIR cells probably reflected the postsynaptic action of GLU, which was released on a similar
time scale. At the end of the experiment, the level
of GLU was still high, that of DA was decreasing
but still higher compared to the controls. The number of c-fosIR cells displayed maximum at 1 h and a
gradual decrease thereafter. We think that this was indicating the primary importance of extracellular GLU
in the initiation and maintenance of c-fos expression. On the other hand, GABA, DA, NE, and 5-HT
could play role in the termination of the c-fos gene
expression by means of inhibition of neuronal activity and the closure of the voltage-dependent Ca2+
channels.
Because of the relatively rapid decrease of Fos immunoreactivity in the striatum, we have to consider
Fos degradation, too. Fos protein is unstable and is
presumably degraded by the proteasome (Acquaviva
et al., 2002). The mechanisms are not clear, but it
seems, that extracellular signals regulate not only the
transcription of the c-fos gene, but also the translocation, viability, and regulatory activity of the Fos protein
in the cell nucleus (Acquaviva et al., 2002). The striatum presents an excellent model for the further study
the role and significance of these extracellular signals,
because the neuronal populations can be identified immunohistochemically, and with double immunostaining methods (Mihály et al., 2001) the localization of
Fos protein in separate neuronal types can be studied
in detail.
4.7. Seizure-related dynamics of c-fos expression
Acknowledgements
Our findings concerning the appearance of c-fos in
the convulsing striatum are in accord with literature
data on seizure-induced expression of ITF’s (Gass
et al., 1992; Willoughby et al., 1995). Detection and
evaluation of Fos immunoreactivity appears suitable
for the histological mapping of epileptic neuronal
The work was supported by the Hungarian National Research Fund (OTKA T 32566, T 037224,
and T/11029131). The technical support from Mrs.
Mónika Herczegh-Kara, Mrs. Márta Dukai, Mrs.
Katalin Lakatos, and Mrs. Andrea Imre-Kobolák is
greatly appreciated.
128
A. Kovács et al. / Epilepsy Research 55 (2003) 117–129
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