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Neurología. 2010;25(3):148-155
NEUROLOGÍA
www.elsevier.es/ neurologia
ORIGINAL ARTICLE
Understanding the pathophysiology of epilepsy in an animal
model: pentylenetetrazole induces activation but not death
of neurons of the medial extended amygdala
G. Pereno * and C. Beltramino
Cát edra de Neurología y Psicología, Facult ad de Psicología, Universidad Nacional de Córdoba, Argent ina
Received on 10t h June 2009; accept ed on 20t h January 2010
KEYWORDS
Medial ext ended
amygdala;
Epilepsy;
Fos;
GABA;
Pent ylent et razole
Abstract
Int roduct ion: Since middle of t he 20t h cent ury t he import ance of amygdala in epilepsy it
has suggest ed, alt hough t he basic mechanisms of t his part icipat ion are st ill unknown.
This ignorance increases when t he different subdivisions of amygdala are considered,
especially t he medial amygdala. In t his work we assess t he involvement of t he medial
ext ended amygdala in an animal model of epilepsy and t he consequences of it s applicat ion
in t his brain st ruct ure.
Mat erial and met hods: Fort y eight adult Wist ar male rat s were used, of which 24 of t hem
received i.p. inj ect ions of pent ylenet et razole, and 24 (cont rols) were inj ect ed wit h
saline. Aft er 2, 6, 12 and 24 h survival, animals were fixed; t he brains were sect ioned
serially and st ained for fos (immunochemist ry) and for neuronal deat h wit h t he A-Cu-Ag
t echnique. Dat a were analysed using t wo-way ANOVA followed by t he Fisher post hoc
t est .
Result s: Very few or no fos-immunoreact ive neurons were seen in cont rol animals. In
experiment al animals, fos was rapidly induced in st ruct ures of medial ext ended amygdala
wit h peak levels at 2 h. Marked fos immunoreact ivit y persist ed up t o 12 h followed by a
gradual ret urn t o baseline at 24 h. However, st at us epilept icus did not induced neuronal
deat h.
Conclusions: These result s show involvement of medial ext ended amygdala in epilept ic
mechanisms wit h an inhibit ory component . However, neuronal deat h is not a consequence
of st at us epilept icus-induced by pent ylent et razole.
© 2009 Sociedad Española de Neurología. Published by Elsevier España, S.L. All right s
reserved.
* Aut hor for correspondence.
E-mail: [email protected] (G. Pereno).
0213-4853X/ $ - see front mat t er © 2009 Sociedad Española de Neurología. Published by Elsevier España, S.L. All right s reserved.
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Pat hophysiology of epilepsy in an animal model
PALABRAS CLAVE
Amígdala ext endida
medial;
Epilepsia;
Fos;
GABA;
Pent ilent et razol
149
Descifrando la fisiopatología de la epilepsia en un modelo animal: el pentilentetrazol
induce la activación pero no la muerte de las neuronas de la amígdala extendida
medial
Resumen
Int roducción: Desde mit ad del siglo XX se ha apunt ado a la import ancia de la amígdala en
la epilepsia, aunque los mecanismos básicos de est a part icipación en su mayoría son aún
desconocidos. Est a ignorancia es aún mayor cuando se t ienen en cuent a las dist int as
subdivisiones de la amígdala, especialment e sus part es mediales. En est e t rabaj o evaluamos la part icipación de la amígdala ext endida medial en un modelo animal de epilepsia, así como las consecuencias que t iene el epilept ógeno en est a est ruct ura cerebral.
Mat erial y mét odos: Se ut ilizaron rat as adult as Wist ar machos (n = 48); 24 animales recibieron inyecciones int raperit oneales de pent ilent et razol y 24, de salina. Luego de 2,
6, 12 y 24 h de sobrevida, los animales se fij aron, y sus cerebros se cort aron seriadament e y se procesaron para fos (inmunoquímica) y muert e neuronal con la t écnica A-Cu-Ag.
Los dat os se analizaron con un ANOVA de 2 vías seguido de un t est post -hoc (LSD de
Fisher).
Result ados: Muy poca act ivación fos se halla en animales cont roles. En animales experiment ales, fos fue rápidament e inducida en la amígdala ext endida medial a las 2 h. Est a
act ivación fue sost enida hast a las 12 h y ret ornó a valores basales a las 24 h. Sin embargo,
el est ado epilépt ico no produj o muert e neuronal.
Conclusiones: Se demuest ra así una part icipación de la amígdala ext endida medial en
mecanismos epilépt icos en los cuales subyace un component e inhibit orio. Sin embargo,
el est ado epilépt ico inducido no produce muert e neuronal en est a est ruct ura.
© 2009 Sociedad Española de Neurología. Publicado por Elsevier España, S.L. Todos los
derechos reservados.
Introduction
Knowledge of t he cellular and molecular mechanisms
underlying t he different t ypes of epilepsy is st ill limit ed.
Current ly, t he fact ors t hat t ransform a normal region of t he
brain int o an epilept ic one are unknown, so st udying animals
wit h epilepsy is very useful for underst anding t he
pat hophysiology of t his group of diseases1.
The most commonly used animal model in st udying
epilepsy is t he model of st at us epilept icus (SE). Different
convulsive agent s are used t o provoke SE, such as kainic
acid2, pilocarpine3 and pent ylenet et razole (PTZ) 4 t o induce
an acut e phase of SE charact erised by unremit t ing t onicclonic crises. The basic problem in experiment al epilept ology
is t o det ermine t he condit ions t hat affect t he balance
bet ween neuronal excit at ion and inhibit ion in each case.
Furt hermore, t he anat omical st ruct ure called t he medial
ext ended amygdala (MeXAM) developed by our laborat ory5 is
an area of t he brain t hat has not been st udied in dept h in t he
field of experiment al epilepsy. It consist s of t wo maj or
subdivisions, one t hat is t he cent ral amygdala and anot her
t hat includes t he medial amygdala and t he int erst it ial nucleus
of t he medial st ria t erminalis (INSTm), known as t he medial
division. The MeXAM syst em is involved in pheromone signal
processing, wit h effect s on t he endocrine syst em and t he
emot ional and reproduct ive aspect s of behaviour 6,7.
Numerous report s involve t he amygdaloid complex in
diseases such as epilepsy8, Alzheimer’s disease9, depression10
and anxiet y11. Specifically in t he field of epilepsy, int erest in
t he amygdala as an epilepsy-relat ed st ruct ure emerged in
t he decade of t he fift ies, when significant neuronal damage
was found in pat ient s who experienced SE. Animal models
have shown t hat t he amygdala is likely t o be damaged in
st at us epilept icus and t hat t his causes hist opat hological
changes12,13. However, very lit t le is known about t he det ails
of t emporal changes and dist ribut ion of t hese MeXAM
changes.
One met hod of evaluat ing t hese changes is t hrough t he
st udy of early gene expression14. Since it s discovery as an
early t ranscript ion gene, c-f os has been used as a t ool f or
f unct ional and anat omical mapping because it ident ifi es
neural cells and circuit s t hat are act ivat ed in response t o
dif f erent st imuli 14. In t his st udy, we at t empt ed t o reveal
t he act ivat ion pat t ern wit h t he expression of t he c-f os
gene, t he f os prot ein, af t er inducing SE wit h PTZ.
Similarly, we conduct ed a st udy t o reveal possible
neuronal deat h as an ef f ect of t he applicat ion of t he
epilept ogenic agent . The agent PTZ has been used
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150
f requent ly in animals as a model of generalised epilepsy;
binding t o t he picrot oxin sit e f or t he GABAA recept or, PTZ
damages chloride channel act ivit y and t hus blocks GABAmediat ed inhibit ion15.
Material and methods
Scope of the study
This st udy was carried out at t he Laborat ory of Limbic Syst em
Neurobiology at t he Inst it ut o de Invest igaciones Médicas
Mercedes y Mart ín Ferreyra.
Study period
The experiment al work st art ed in February 2006 and ended
aft er 12 mont hs, in March 2007.
Animals
We used 48 adult male Wist ar rat s (200-250 g), isolat ed in
ad-hoc housing 48 h before t he experiment in general condit ions for animal facilit ies. All procedures were performed
in accordance wit h Royal Decree 1201/ 2005 on t he prot ect ion of experiment al animals.
Induction of status epilepticus with PTZ
The animals (n=6) were inj ect ed wit h int raperit oneal PTZ
dilut ed in 0.9% saline solut ion according t o t he scheme of
Pineau et al.16. This st udy considered only t he animals which
reached st at e 4. In parallel, cont rol animals received saline
solut ion in t he same manner.
Fixation of the brain
At 2, 6, 12 and 24 h aft er inducing SE, t he animals were
anaest het ised wit h chloral hydrat e at 30%, t hen infused wit h
100 ml of washing solut ion and finally 300 ml of 4%
paraformaldehyde. The brain was t hen placed in sucrose
solut ion at 30%; 48 h lat er it was cut in 40 μm slices on a
freezing microt ome and collect ed serially in t he same fixat ive
solut ion.
Immunochemical technique
For fos prot ein det ect ion, t he brain sect ions were washed in
buffer (PO4, 0.01 mol) and t he int ernal peroxidase was
inhibit ed. The sect ions were washed and t he non-specific
bindings were blocked and t hen incubat ed in normal horse
serum at 5%. Lat er, t he sect ions were incubat ed in primary
ant ibody (fos: 1/ 5,000, sc-52, Sant a Cruz) and t hen incubat ed
for 2 h in secondary biot inylat ed ant ibody (1/ 200, Vect or)
and 2 h wit h avidin-biot in-peroxidase complex (Vect or); t he
react ion was developed wit h diaminobenzidine as t he
chromogen, adding cobalt chloride (black react ion product ).
The sect ions were mount ed and coverslipped wit h DPX.
G. Pereno et al
Amino-cupric-silver technique
The met hod of impregnat ion wit h A-Cu-Ag17 was select ed
because it is usually applied in t he laborat ory and is t he
lat est , improved version of copper-silver met hods.
Quantitative cell analysis
The sect ions were analysed wit h an Axioplan microscope,
wit h an adapt ed Leica camera. The quant it at ive cell analysis
was performed using Scion Image soft ware (Scion
Corporat ion, 2000, NIH Image, USA) in coronal sect ions
increased by x20.
We select ed t he following MeXAM st ruct ures18 for analysis:
medial amygdaloid nucleus (Me) in t he planes relat ive t o
bregma: —1.80 and —2.56 for t he ant erodorsal Me (MeAD),
—2.30 for t he ant erovent ral Me (MeAV), —3.14 and —3.60
for t he post erodorsal Me (MePD) and —3.14 for t he
post erovent ral Me (MePV); t he INSTm in t wo planes relat ive
t o bregma: —0.30 for t he ant erior (INSTa) and vent ral
(INSTv) INST and —0.80 for t he post erior (INSTp).
Statistical analysis
The dat a were analysed using a t wo-fact or analysis of
variance (ANOVA), followed by Fisher’s post -hoc least
significant difference (LSD) t est . The values were expressed
as t he average of each group±st andard error, and p <0.05
was considered as t he limit of st at ist ical significance.
Results
Expression of fos after PTZ injection
In all cont rol animals inj ect ed wit h saline solut ion, few,
scat t ered nuclei of cells, act ivat ed in t he areas evaluat ed
here, were observed.
In experiment al animals, t he highest fos prot ein
expression was found in t hose who survived 2 h aft er
inj ect ion, where t he differences wit h t he cont rol animals
were st at ist ically significant for all t he areas where
quant it at ive cell analysis of fos (+) nuclei was carried out
(t ables 1 and 2). The MeAD (t able 1) and t he INSTp (t able 2)
were t he areas where it was possible t o observe t he great est
act ivat ion compared wit h cont rols. Likewise, t he MePD (fig.
1) and t he INSTa (fig. 2) were bot h heavily marked for fos
prot ein. In all areas under evaluat ion, t here were numerous
cell nuclei st ained wit h fos ant ibody. Three point s st and out
especially: t he different iat ion of t he t hree columns in t he
INSTp (5), a considerable amount of act ivat ed cells in all Me
divisions in t he region near t he accessory olfact ory t ract
and t he opt ic t ract (fig. 1), and t he INSTv (fig. 2) below t he
ant erior edge.
Aft er a survival of 6 h, alt hough marking decreased, t he
differences remained significant , as was also t rue in t he
animals sacrificed 12 h aft er PTZ inj ect ion. Once again, t he
previously ment ioned areas were t he ones wit h t he great est
act ivat ion (figs. 1 and 2; t ables 1 and 2.) Finally, in animals
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Pat hophysiology of epilepsy in an animal model
151
Table 1 Quant it at ive analysis of immunoreact ive nuclei for fos in medial amygdaloid nucleus subdivisions aft er
pent ylenet et razole (PTZ)-induced st at us epilept icus (SE)
St ruct ure
MeAV
Survival aft er PTZ-induced SE
Cont rol
2h
6h
6 ± 1.02
27.6 ± 2.43a
12 h
17.3 ± 1.66a
24 h
8.6 ± 2.44
5.3 ± 1.18
MeAD —1.80
16.1 ± 1.03
158.6 ± 7.71
97.6 ± 5.5
53.8 ± 3.01
20.83 ± 1.86
MeAD —2.56
19 ± 1.2
202.5 ± 4.09a
102 ± 5.43a
49.16 ± 2.25a
23.8 ± 2.04
MePV
14.5 ± 1.49
111.5 ± 4.21a
98.33 ± 3.56a
48.3 ± 1.28a
22 ± 1.26b
MePD —3.14
16.1 ± 1.03
165.8 ± 7.03a
102.6 ± 4.86a
57 ± 2.08a
20 ± 1.96
MePD —3.60
19 ± 1.2
a
a
a
a
a
192 ± 4.86
a
100.1 ± 4.36
47.8 ± 3.32
23.3 ± 2.33
MeAD: ant erodorsal medial amygdaloid nucleus; MeAV: ant erovent ral medial amygdaloid nucleus; MePD: post erodorsal
medial amygdaloid nucleus; MePV: post erovent ral medial amygdaloid nucleus.
We used a t wo-fact or ANOVA followed by Fisher’s post -hoc LSD t est . The dat a are expressed as average ± st andard error of
t he average.
a
b
p<0.001.
p<0.01.
Table 2 Quant it at ive analysis of t he immunoreact ive nuclei for fos in medial INST subdivisions aft er pent ylenet et razole
(PTZ)-induced st at us epilept icus (SE)
St ruct ure
INSTa
INSTp
INSTv
Survival aft er PTZ-induced SE
Cont rol
2h
6h
12 h
24 h
13.6 ± 1.59
18.3 ± 1.08
12.1 ± 1.48
167.6 ± 6.4*
207.5 ± 8.42*
84.16 ± 3.51*
126.5 ± 9.68*
132.6 ± 5.68*
62.5 ± 2.26*
50.1 ± 5.32 *
55.5 ± 3.97 *
45 ± 2.5 *
16.5 ± 1.46
21.5 ± 2.87
11.16 ± 0.91
INST: int erst it ial nucleus of t he st ria t erminalis; INSTa: int erst it ial nucleus of t he ant erior st ria t erminalis; INSTp: int erst it ial nucleus
of t he post erior st ria t erminalis; INSTv: int erst it ial nucleus of t he vent ral st ria t erminalis.
We used a t wo-fact or ANOVA followed by Fisher’s post -hoc LSD t est . The dat a are expressed as average±st andard error of t he
average.
* p<0.001.
Figure 1 Phot ographs of brain sect ions (?2.5) showing t he
act ivat ion in t he post erodorsal and post erovent ral medial
amygdaloid nuclei at different survivals aft er pent ylenet et razole
inj ect ion. Bar scale: 500 μm.
Figure 2 Phot ographs of brain sect ions (× 2.5) showing t he
act ivat ion in t he int erst it ial nucleus of t he ant erior and vent ral
st ria t erminalis at different survivals aft er pent ylenet et razole
inj ect ion. Bar scale: 500 μm.
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152
G. Pereno et al
Neurodegeneration after PTZ injection
Under the paradigm of this model, it was not possible to find
neuronal death in the MeXAM (fig. 5). While animals were used
with survival up to 24 h, animals with survival rates of up to 30
days after PTZ inj ection were also used and no signs of neuronal
death were found in any of them. However, it should be noted
that it was possible to find signs of axon terminal
neurodegeneration in the CA3 area in the hippocampus of
animals with 24-h survival (results not shown). This feature
was not observed in other brain structures.
200
NIETa (–0.30)
180
MePD (–3.14)
180
Control
Experimental
*
160
No. of fos cells (+)
140
*
140
120
100
80
60
*
40
20
0
200
Control
Experimental
*
160
No. of fos cells (+)
whose survival reached 24 h, it could be observed t hat t he
differences bet ween cont rol and experiment al animals
were not significant , as act ivat ion reached baseline values
comparable t o t hose of cont rols (t ables 1 and 2).
The ANOVA performed revealed what was st at ed
previously, t hat is, t hat t he act ivat ion peak was found at 2
h and marking t hen gradually decreased in animals wit h
longer survival (figs. 3 and 4). The fact t hat t he differences
bet ween t he various experiment al groups were significant
for most of t he st ruct ures in t he quant it at ive cell analysis
(t ables 1 and 2) is not ewort hy.
2h
6h
12 h
24 h
Figure 4 Quant it at ive cell analysis of fos+ nuclei in animals
wit h survivals of 2, 6, 12 and 24 h aft er pent ylenet et razole
applicat ion and subsequent t wo-fact or ANOVA in t he int erst it ial
nucleus of t he ant erior st ria t erminalis (INSTa), whose fact ors
are t he “ t reat ment ” and t he various “ survivals” . The result of
t he st udy of t he fact ors is as follows: for t reat ment :
F(1.40)=640.46; p<0.001; for survival: F(3.40)=130.34; p <0.001; int eract ion: F(3.40)=132.90; p<0.001. The result s are expressed as
average±st andard error (* p<0.001).
120
*
100
80
*
60
40
20
0
2h
6h
12 h
24 h
Figure 3 Quant it at ive cell analysis of fos+ nuclei of animals
wit h survivals of 2, 6, 12 and 24 h aft er pent ylenet et razole
applicat ion and subsequent t wo-fact or ANOVA in t he post erodorsal medial amygdaloid nucleus (MePD), whose fact ors are
t he “ t reat ment ” and t he various “ survivals” . The result of t he
st udy of t he fact ors is as follows: for t reat ment : F(1.40)=1,111;
p<0.001; for survival: F(3.40)=220.3; p <0.001; int eract ion:
F(3.40)=209.4; p<0.001. The result s are expressed as
average±st andard error (*p<0.001).
Figure 5 Phot ographs of brain sect ions (× 2.5) showing in A t he
post erodorsal and post erovent ral medial amygdaloid nucleus,
and in B t he int erst it ial nucleus of t he ant erior and vent ral st ria
t erminalis of animals wit h survival 24 h aft er st at us epilept icus
and which were processed wit h t he A-Cu-Ag t echnique.
Discussion
GABAergic neurotransmission and epilepsy
Several findings demonst rat e t hat t he GABAergic syst em is
involved in various t ypes of epilepsies. For example, Muñoz
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Pat hophysiology of epilepsy in an animal model
et al. 19, when analyzing t he brains of epilept ic pat ient s,
found a reduct ion in GABAB recept or 1a-b immunost aining
in t he granular cell layer of t he hippocampus dent at e gyrus.
Ot her aut hors found t he t wo recept or GABAB subt ypes (1
and 2) overexpressed in t he hippocampus of pat ient s wit h
t emporal lobe epilepsy, which was int erpret ed as a
compensat ing mechanism20. The values of t he GABAA
recept or were also alt ered in t he brains of epilept ic pat ient s
and animal models21.
GABA is t hus implicat ed in epilepsy, st at us epilept icus
and ot her epilept ic syndromes. Presynapt ic and post synapt ic
alt erat ions, such as changes in recept or subunit composit ion
and recept or st ruct ure or genet ic modificat ions of recept ors,
could affect suscept ibilit y t o an epilept ic seizure.
However, epilepsy is not a GABAergic syndrome. Several
findings led t o t he idea t hat seizures would respond t o a
simple model in which inhibit ion and excit at ion would act
as brake and accelerat or, respect ively. This scheme is
nevert heless an oversimplified model. There is evidence
t hat GABAergic neurot ransmission can be excit at ory in
baseline condit ions not only in a developing brain22, but also
in an epilept ic brain23.
In summary, an increase in t he number of GABAergic
synapses has been found in human epilept ic brains and in
animal models. These have been int erpret ed as a
compensat ory mechanism, but t hey could also be explained
as being secondary t o GABAergic funct ion changes (from
inhibit ion t o excit at ion) and being, part ially, t he origins of
syst em hyperexcit abilit y1.
Expression of early genes and epilepsy
Previous st udies have used fos prot ein t o assess t he
involvement of different neuronal populat ions in epilepsy
models using PTZ24,25. Here we show, aft er t he quant it at ive
analysis of fos-act ivat ed neuron nuclei, t hat expression is
maximal at 2- and 4-h survival, being consist ent wit h acut e
expression, alt hough t here is evidence of sust ained
expression unt il 12 h aft er t he SE. Aft er 12 h, immunost aining
decreases considerably; t hat is why, in t his st udy, it was
decided not t o sacrifice animals surviving over 24 h aft er
inj ect ion.
Taking fos prot ein expression as a t ool for funct ional and
anat omical mapping, we show t he abilit y of t he MeXAM t o
react by becoming act ive t hrough t he expression of early
genes (such as c-fos) and it s part icipat ion in epilept ic
mechanisms. This let s us st at e t hat t he MeXAM is involved
in epilept ic processes involving t he part icipat ion of
GABAergic mechanisms.
The fact t hat fos expression was evident at 24 h point ed
t o t he hypot hesis t hat t hese neurons might be suffering
from some form of neurodegenerat ive process. To t his end,
parallel sect ions of brains were processed wit h t he A-Cu-Ag
t echnique. Despit e using different PTZ doses (from mild
seizures t o severe SE), no degenerat ion was found in t hese
brains. This shows t hat , while t he MeXAM is involved in t his
animal model, t he neurons were able t o recover from t he
st ress represent ed by PTZ-induced SE.
153
The involvement of the medial extended amygdala
in epileptic mechanisms
The amygdaloid complex has been lit t le st udied in t he field
of epilepsy, despit e t he fact t hat it s involvement has been
demonst rat ed, part icularly t hat of t he t emporal lobe26.
There are basic research st udies t hat have addressed t he
problem of epilepsy using different animal models. They
emphasise t he role of t he hippocampus and of some
amygdaloid nuclei of t he basolat eral complex, leaving aside
ot her amygdaloid complexes. Given t he above, t he quest ion
arises: is t he MeXAM really involved in epilepsy? The result s
of our st udy show t hat t he MeXAM clearly part icipat es in
epilept ic mechanisms.
Alt hough t he use of early gene product s, such as c-fos, as
a marker of neural act ivit y revealed t hat ot her brain areas
were also act ivat ed, t here are t wo import ant point s t o
consider:
1. The animal model used here corresponded t o secondary
generalised epilepsy and it t herefore also act ivat ed ot her
brain areas.
2. In t hese areas, fos expression decreased rapidly (at about
4 h), whereas fos expression was maint ained significant ly,
unt il 12 h of survival, in MeXAM st ruct ures.
The result s obt ained here demonst rat e t hat t he MeXAM is
involved in epilept ic mechanisms. However, unlike what
happens wit h ot her animal models such as kainic acid27 or
pilocarpine28, no neuronal deat h was found in t he MeXAM. It
has been report ed t hat syst emic PTZ inj ect ion induces
neurodegenerat ion in t he neurons of t he hippocampus and
t he amygdala29; however, t he neurodegenerat ion found in
t hese st udies was apopt ot ic, which cannot be det ect ed wit h
t he t echnique used in t his st udy. In addit ion, our st udy
considers t he amygdala as a unit ary complex; it does not
discriminat e t he different subdivisions. Likewise, it has also
been demonst rat ed t hat PTZ-induced SE brings about
oxidat ive st ress in rat brains, wit h consequent neuronal
deat h30. The t echnique used here is specifically for det ect ing
neuronal deat h from necrosis and t he deat h of neurons
induced by oxidat ive st ress very probably involves an
apopt ot ic component 31.
Ot her st udies16 have shown t hat PTZ-induced st at us
epilept icus in immat ure rat s causes a period of st rong
act ivat ion charact erised by a sharp increase in met abolic
rat e; t his has t he effect , at mat urit y, of permanent changes
in cell membrane permeabilit y, allowing acid fuchsin t o
ent er. However, t hese cells never ent er t he st age of DNA
fragment at ion and are able t o recover.
The cont radict ory result s might be due t o using dif f erent
t echniques f or det ect ing neuronal deat h, or t o considering
t he amygdaloid complex as a whole wit hout t aking it s
various subdivisions int o account . Our findings demonst rat e
t hat t he GABAergic ant agonist , PTZ, does not cause
neuronal deat h in t he MeXAM, which shows t hat epilepsies
in which t he underlying cause is most ly an inhibit ory
component are unlikely t o result in t he deat h of it s
neurons.
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154
G. Pereno et al
Clinical projections and research practices
While t his st udy has a dist inct ly basic orient at ion, it enables
import ant conclusions t o be drawn. First , t his research
provides a basis for underst anding t he complexit y of t he
amygdaloid complex. This is a brain st ruct ure t hat , despit e
it s small size, has great complexit y, bot h in it s funct ions
and it s anat omy, in t erms of it s connect ions wit h ot her
st ruct ures.
This st udy is t he st art ing point for furt her basic and
clinical research. In t he basic aspect , it is import ant for t he
development of new ant iepilept ic drugs, GABAergic
ant agonist s or agonist s, which int erfere wit h t he
epilept ogenic process. In t he clinical aspect , it est ablishes
a correlat ion bet ween t he present research wit h resist ant
t emporal lobe epilepsy and t he choice of different drugs for
it s t reat ment .
Det ailed knowledge of t he anat omy of t he amygdala will
facilit at e, in t he near fut ure, select ive surgery of t emporal
lobe epilepsy. Of course, all of t he preceding point s lie
wit hin t he framework of basic research effort s, in const ant
int eract ion wit h clinical research, aimed at offering new
init iat ives t hat provide a deeper underst anding of t his
disease.
Financing
This st udy received funding from t he Secret ariat for
Science and Technology of t he Universidad Nacional de Cordoba.
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Conflict of interests
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The aut hors declare no confl ict of int erest s.
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References
21.
1. De la Vega C, Villanueva-Hernandez P, Priet o-Mart ín A.
Neuroquímica de la epilepsia, neurot ransmisión inhibit oria y
modelos experiment ales: nuevas perspect ivas. Rev Neurol.
2006;42:159-68.
2. Ben-Ari Y. Limbic seizure and brain damage produced by kainic
acid: Mechanisms and relevance t o human t emporal lobe
epilepsy. Neuroscience. 1985;14:375-403.
3. Curia G, Longo D, Biagini G, Jones R, Avolia M. The pilocarpine
model of t emporal lobe epilepsy. J Neurosci Met hods. 2008;172:
143-57.
4. It oh K, Wat anabe M. Paradoxical facilit at ion of PTZ-induced
convulsion suscept ibilit y in mice lacking neuronal nit ric oxide
synt hase. Neuroscience. 2009;159:735-43.
5. De Olmos JS, Belt ramino CA, Aldheid, G. Amygdala and
ext ended amygdala of t he rat : A Cyt oarchit ect onical,
Fibroarchit ec-t onical, and Chemoarchit ect onical Survey. In:
Paxinos G, edit or. The rat nervous syst em. 3rd ed. Sydney:
Elsevier; 2004. p. 509-603.
6. Keverne EB, De la Riva C. Pheromones in mice: Reciprocal
int eract ions bet ween t he nose and brain. Nat ure. 1982;296:
148-50.
7. Belt ramino CA, Taleisnik S. Release of LH in t he female rat by
olfact ory st imuli. Effect of removal of t he vomeronasal organ
22.
23.
24.
25.
26.
27.
or lesioning of t he accessory olfact ory bulbs. Neuroendocrinol.
1983;36:53-8.
Pit känen A, Tuunanen J, Kalviainen R, Part anen, K, Salmenpera
T. Amygdala damage in experiment al and human t emporal lobe
epilepsy. Epilepsy Res. 1998;32:233-53.
Mart ínez-Cast illo E, Arrazola J, Fernández A, Maest ú F, Ort iz T.
At rofia del complej o amigdalino y expresión neuropsiquiát rica
de la enfermedad de Alzheimer. Rev Neurol. 2001;33:477-82.
Nest ler EJ, Barrot M, DiLeone RJ, Eisch AJ, Gold SJ, Mont eggia,
LM. Neurobiology of depression. Neuron. 2002;34:13-25.
St ein MB, Simmons AN, Feinst ein JS, Paulus MP. Increased
Amygdala and insula act ivat ion during emot ion processing in
anxiet y-prone subj ect s. Am J Psychiat ry. 2007;164:318-27.
Ding M, Haglid KG, Hamberger A. Quant it at ive immunochemist ry
on neuronal loss, react ive gliosis and BBB damage in cort ex/
st riat um and hippocampus/ amygdala aft er syst emic kainic acid
administ rat ion. Neurochem Int . 2000;36:313-8.
Riba-Bosch A, Pérez-Clausell J. Reponse t o kainic acid
inj ect ions: changes in st aining for Zinc, fos, cell deat h and glial
response in t he rat forebrain. Neuroscience. 2004;125:803-18.
Kovacs K. C-fos as a t ranscript ion fact or: a st ressful (re) view
from a funct ional map. Neurochem Int . 1998;33:287-97.
André V, Pineau N, Mot t e JE, Marescaux C, Nehlig A. Mapping of
neuronal net works underlying generalized seizures induced by
increase doses of PTZ in t he immat ure and adult rat : a c-Fos
immunohist ochemical st udy. Eu J Neurosci. 1998;10:2094-106.
Pineau N, Charriaut -Marlangue C, Mot t e J, Nehlig A. PTZ
seizures induce cell suffering but not deat h in t he immat ure
rat brain. Brain Res Dev Brain Res. 1999;112:139-44.
De Olmos JS, Belt ramino CA, De Olmos de Lorenzo S. Use of an
amino-cupric-silver t echnique for t he det ect ion of early and
semiacut e neuronal degenerat ion caused by neurot oxicant s,
hypoxia, and physical t rauma. Neurot oxicol Terat ol. 1994;16:
545-61.
Paxinos G, Wat son C. The rat brain in st ereot axic coordinat es.
Sidney: Academic Press; 1995.
Muñoz A, Arellano JI, Defelipe J. GABABR1 recept or prot ein
expresión in human mesial t emporal cort ex: changes in
t emporal lobe epilepsy. J Comp Neurol. 2002;449:166-79.
Furt inger S, Pirker S, Czech T, Baumgart ner C. Increased
expression of gamma aminobut yric acid t ype B recept ors in t he
hippocampus of pat ient s wit h t emporal lobe epilepsy. Neurosci
Let t . 2003;352:141-5.
Schawarzer C, Tsunanshima K, Fuchs K, Sleghart W. GABAA
recept or subunit s in t he rat hippocampus II: alt ered dist ribut ion
in kainic acid-induced t emporal lobe epilepsy. Neuroscience.
1997;80:1001-17.
Ben-Ari Y. Excit at ory act ions of GABA during development : t he
nat ure of t he nurt ure. Nat Rev Neurosci. 2002;3:728-39.
St raessie A, Loup F, Arabadzisz D, Ohning GV, Frit schy JM. Rapid
and long-t erm alt erat ions of hippocampal GABAB recept ors in
a mouse model of t emporal lobe epilepsy. Eu J Neurosci. 2003;
18:2213-26.
Baraban SC, Taylor MR, Cast ro PA, Baier H. PTZ induced changes
in zebrafish behavior, neural act ivit y and c-fos expression.
Neuroscience. 2005;131:759-68.
Erdt mann-Vourliot is M, Riechert U, Mayer P, Grecksch G, Höllt
V. PTZ-induced c-fos expression in t he hippocampus of kindled
rat s is suppressed by concomit ant t reat ment wit h naloxone.
Brain Res. 1998;792:299-308.
Past or J, Uzcát egui Y, Gal-Iglesias B, Ort ega G, Sola R, Menéndez
de la Prida L. Bases fisiopat ológicas de la epilepsia del lóbulo
t emporal: est udios en humanos y animales. Rev Neurol. 2006;
42:663-73.
Miyamot o R, Shimakawa S, Susuki S, Ogihara T, Tamai H.
Edaravone prevent s kainic acid-induced neuronal deat h. Brain
Res. 2008;1209:85-91.
Document downloaded from http://www.elsevier.es, day 18/06/2017. This copy is for personal use. Any transmission of this document by any media or format is strictly prohibited.
Pat hophysiology of epilepsy in an animal model
28. Liu YH, Wang L, Wei LC, Huang YG, Chen LW. Up-regulat ion of
D: —serine Might Induce GABAergic neuronal degenerat ion in
t he cerebral cort ex and hippocampus in t he mouse pilocarpine
model of epilepsy. Neurochem Res. 2009;34:1209-18.
29. Park JH, Cho H, Kim H, Kim K. Repeat ed brief epilept ic seizures
by PTZ cause neurodegenerat ion and promot e neurogenesis in
discret e brain regions of freely moving adult rat s. Neuroscience.
2006;140:673-84.
155
30. Obay BD, Tasdemir E, Tümer C, Bilgin HM, At maca M. Dose
dependent effect s of ghrelin on pent ylenet et razole-induced
oxidat ive st ress in a rat seizure model. Pept ides. 2008;29:
448-55.
31. Pellegrini M, Baldari CT. Apopt osis and oxidat ive st ress-relat ed
diseases: t he p66Shc connect ion. Curr Mol Med. 2009;9:
392-8.