Antiepileptic effect of acylpolyaminetoxin JSTX

Brain Research 1048 (2005) 170 – 176
www.elsevier.com/locate/brainres
Research report
Antiepileptic effect of acylpolyaminetoxin JSTX-3 on rat
hippocampal CA1 neurons in vitro
Simone Denise Salamonia, Jaderson Costa da Costaa,*, Mário Sérgio Palmab,d,
Katsuhiro Konnoc,d, Ken-ichi Niheib,d, Andréa Alencar Tavaresa, Daniela Souza de Abreua,
Gianina Teribele Venturina, Fernanda de Borba Cunhaa,
Raquel Mattos de Oliveiraa, Ricardo Vaz Bredaa
a
Laboratório de Neurociências, Instituto de Pesquisas Biomédicas, Pontifı́cia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brasil
b
Laboratório de Biologia Estrutural, CEIS/Departamento de Biologia, Instituto de Biociências, UNESP, Rio Claro, SP, Brasil
c
Instituto Butantan, São Paulo, SP, Brasil
d
CAT/CEPID-FAPESP, Brasil
Accepted 22 April 2005
Available online 23 May 2005
Abstract
The Joro spider toxin (JSTX-3), derived from Nephila clavata, has been found to block glutamate excitatory activity. Epilepsy has been
studied in vitro, mostly on rat hippocampus, through brain slices techniques. The aim of this study is to verify the effect of the JSTX-3 on the
epileptiform activity induced by magnesium-free medium in rat CA1 hippocampal neurons. Experiments were performed on hippocampus
slices of control and pilocarpine-treated Wistar rats, prepared and maintained in vitro. Epileptiform activity was induced through omission of
magnesium from the artificial cerebrospinal fluid (0-Mg2+ ACSF) superfusate and iontophoretic application of N-methyl-d-aspartate
(NMDA). Intracellular recordings were obtained from CA1 pyramidal neurons both of control and epileptic rats. Passive membrane
properties were analyzed before and after perfusion with the 0-Mg2+ ACSF and the application of toxin JSTX-3. During the ictal-like activity,
the toxin JSTX-3 was applied by pressure ejection, abolishing this activity. This effect was completely reversed during the washout period
when the slices were formerly perfused with artificial cerebrospinal fluid (ACSF) and again with 0-Mg2+ ACSF. Our results suggest that the
toxin JSTX-3 is a potent blocker of induced epileptiform activity.
D 2005 Elsevier B.V. All rights reserved.
Theme: Disorders of the nervous system
Topic: Epilepsy: anti-convulsant drugs
Keywords: JSTX-3; Pilocarpine; Epilepsy; NMDA; Hippocampus
1. Introduction
Epilepsy is a chronic neurological disorder that affects
0.5 –1% of the population worldwide [38]. The response to
therapy in newly diagnosed cases is generally good, but up
* Corresponding author. Laboratorio Neurociencias, Instituto de Pesquisas
Biomedicas, Hospital Sao Lucas da PUCRS, Av. Ipiranga, 6690. Porto
Alegre, 90.610-000, RS, Brazil.
E-mail address: [email protected] (J. Costa da Costa).
0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2005.04.060
to 30% of patients cannot achieve acceptable seizure
control, despite adequate trials with potentially effective
antiepileptic agents [27]. Furthermore, there is an important
risk for a large number of adverse effects [16]. In order to
develop new antiepileptic therapeutic strategies, it is
fundamental to understand the basic mechanisms involved
in the epileptic discharges. Many diseases and neuronal
disorders are caused by receptor and ion channel dysfunction [19]. Excitatory amino acid receptors represent promising targets for antiepileptic drug development.
S.D. Salamoni et al. / Brain Research 1048 (2005) 170 – 176
Spiders are carnivorous invertebrates that feed on other
Arthropods, mainly insects, and have developed venoms for
this purpose. These venoms represent a great reservoir of
toxins with selective modes of action, able to immobilize or
kill their preys. Neurotoxins are very accurate molecular
tools that selectively activate or block several components
of the mammalian nervous system, including neuroreceptors, neurotransmitter carriers and ion channels. The knowledge about the structure and the mechanisms of action of
different neurotoxins has guided the development of drugs
that could potentially treat neurological diseases [24].
Polyamine toxins, such as the acylpolyaminetoxins from
Nephila clavata [31], are effective blockers of the ion
channels associated with excitatory amino acids receptors.
The action of these toxins depends on the previous bond of
glutamate (pre-activation) to its receptors and, therefore, can
be considered antagonist-toxins, non-competitive for glutamate receptors. Since glutamate receptors are involved in
the pathophysiology of several brain disorders, such as
epilepsy, the blockade of these receptors by acylpolyaminetoxins may have, at least in theory, neuroprotector effects.
In fact, there are reports indicating that these toxins have
anticonvulsant action in several experimental models of
seizure [22].
In most cases, toxins isolated from the venom of spiders
belonging to the genus Nephila are very similar among them
and present a common domain essential for their action on
glutamate receptors [22]. The toxin JSTX-3 was first
described as a component of the venom of the Japanese
spider N. clavata and later also observed in the venom of
Nephilengys cruentata, a spider found in Brazil [32]. Since
then, it has been shown that JSTX-3 is a highly specific
blocker of ionotropic glutamate receptors and, nowadays, it
is consider a very important tool in neurobiology, neurochemistry and neurology research [20,21].
Current experimental models of chronic temporal lobe
epilepsy involve the use of hippocampal slices [2,11,29].
Due to the epileptogenic behavior of the hippocampus and
the fact that its trisynaptic loop utilizes glutamate as main
neurotransmitter, the activity of these pathways is thought to
play a role in epileptogenesis. Actually, perfusion of
hippocampal slices with modified artificial cerebro-spinal
fluid (ACSF) lacking magnesium as a way to overactivate
NMDA receptors (NMDAr) is, at present, one of the widest
employed models to induce and study epileptiform activity
in vitro [3]. At resting membrane potential, the NMDAr
channel becomes blocked by magnesium ions, thus preventing the free passage of other ions through it and
inducing a voltage-dependent inhibition [41]. The mechanism whereby free-magnesium medium elicits ictal-like
activity includes removal of the Mg2+ block of the NMDAr
channel [3]. Although the perfusion of hippocampal slices
with magnesium-free ACSF presents several advantages to
study the electrophysiological properties of the epileptic
discharge, it is obvious that, to fully characterize the
pathophysiology of the status epilepticus, this model has
171
to be used in conjunction with others able to represent an
epileptic event in vivo. In this respect, the induction of
status epilepticus by injection of the cholinergic agonist
pilocarpine in rodents has been utilized to characterize
several aspects of the epileptic brain, ranging from calcium
dynamics [36] to the synthesis and release of neuropeptides
and neuromodulators [13] and the differential expression of
proteins [9,26,39]. It is known that the hyperexcitability
induced by pilocarpine is accompanied by an increase in
glutamate release [10] and that, in turn, this increase may
induce the neuronal death that occurs in several structures,
including the hippocampus, due to status epilepticus [8]. In
fact, it has been reported that the seizures induced by
pilocarpine treatement are mediated by activation of hippocampal NMDAr [40].
Taking into account the above mentioned antecedents,
the aim of the present study was to analyze and characterize
the effect of the acylpolyaminetoxin JSTX-3 on the
epileptiform activity induced by magnesium-free ACSF in
CA1 neurons of hippocampal slices obtined from control
and pilocarpine-treated epileptic rats.
2. Materials and methods
2.1. Experimental groups
Animals (Male Wistar rats, 30– 35 days old) were kept
under controlled temperature on a 12-h light– dark cycle
with food and water ad libitum. Rats were divided into
control and experimental/pilocarpine-injected groups, with 7
animals each. As inclusion criterion in the experimental
group, animals had to achieve degree three in the Racine
Scale, which is considered to represent status epilepticus.
Animals that did not reach degree 3 were not considered
further.
Rats were injected with methylscopolamine (1 mg/kg,
i.p) 30 min before vehicle (saline) or pilocarpine (380 mg/
kg, i.p) administration. Pilocarpine injection triggered limbic
behavior classified according to the Racine Scale [35].
Status epilepticus (SE) is classically considered as the
persistence of continuous seizures for at least 30 min.
Diazepan (4 mg/kg, i.p) was administered 1 h after the onset
of SE to interrupt seizure activity [8]. Rats were given oral
mixture of sucrose and potassium for several days after
pilocarpine-induced SE.
2.2. Brain slices preparation and solutions
Thirty to sixty days after treatment with vehicle or pilocarpine, rats were decapitated under thiopental anesthesia
(40 mg/kg, i.p). Their brains were rapidly removed and
cooled in dissection artificial cerebrospinal fluid (ACSF)
containing (in mM): 130 NaCl, 3.5 KCl, 1.3 NaH2PO4, 5
Mg2+, 0.2 CaCl2, 10 d-glucose and 24 NaHCO3, previously
gassed with a 95% O2 and 5% CO2 mixture to obtain a pH
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S.D. Salamoni et al. / Brain Research 1048 (2005) 170 – 176
value of 7.3 –7.4, before being sectioned coronally and glued
with cyanoacrylate to the stage of a vibratome (Vibroslice
752 M, Campden Instruments, USA). Four hundred micrometers thick coronal hemislices were obtained from the
medial part of the hippocampus and allowed to recover for at
least 1 h before being transferred to a submersion-type
recording chamber and placed on a nylon net submerged in
normal ACSF (in mM 130 NaCl, 3.5 KCl, 1.3 NaH2PO4, 2
Mg2+, 2 CaCl2, 10 d-glucose and 24 NaHCO3, pH 7.4) at
room temperature which was continually gassed with 95%
O2 and 5% CO2. A maximum of two slices per rat was used.
Ictal-like activity and interictal discharges were induced by
either omitting Mg2+ from the oxygenated ACSF (0-Mg2+
ACSF) [3] or iontophoretically applying (20 to 60 nA during
30 s) N-methyl-d-aspartate (NMDA, 25 AM).
2.3. JSTX-3 and drugs
JSTX-3 was synthesized based on Boc-chemistry using
2-nitrobenzenesulfonamide as protecting and activating
group, as previously described [31]. The micropipette
containing JSTX-3 (0,1 AM) diluted in 0-Mg2+ ACSF was
placed next to the intracellular electrode in the CA1
hippocampal region and ejected through a pneumatic pump
(PV830 Pneumatic Pico Pump WPI). N-methyl-d-aspartate
(NMDA, Sigma, St. Louis, MO, USA) was dissolved to
final concentration in Tris-phosphate buffer (pH 8). Microelectrodes were filled with 0.5% biocytin in 0.05 M Tris and
1 M KCl.
2.4. Electrophysiological recordings, data acquisition and
analysis
The stimulation for current clamp as well as the
recording protocols were carried out using an in vitro
electrophysiological system [5], inside a Faraday cage. The
recording electrodes were pulled on a horizontal micropipette puller (Sutter P-87, Sutter Instrument Co., USA)
from borosilicate glass capillaries filled with either 3 M
potassium acetate (electrode impedance 80– 100 MV) or
biocytin in 0.05 M Tris and 1 M KCl (180 – 200 MV) for
intracellular recordings (IC) and ACSF (5 –10 MV) for
extracellular recordings (EC). Recordings were performed
in the target cell on the CA1 hippocampal layer. CyberAmp
380 (Axon Instruments Inc., USA) programmable signal
conditioner was used, and current-clamp recordings were
performed using an Axoclamp-2B amplifier (AxoClamp2B, Axon Instruments Inc., USA). The parameters used to
verify the viability of a neuron were membrane potential,
input resistance and action potential amplitude. Only
neurons with membrane potential of at least 50 mV and
action potentials with amplitude higher than 50 mV were
studied. Furthermore, we studied only slices in which ictallike activity was present after 30 min perfusion with 0-Mg2+
ACSF. Data were monitored and recorded on a personal
computer via the AxoScope software (Axon Instruments)
and analyzed off-line with the Origin 5.0 software (Microcal
Software Inc.). Sample cells were filled with biocytin in
order to identify neuronal morphology.
The electrophysiological protocol consisted of seven
consecutive stages (A –G). (A) Cells were perfused with
normal ACSF (30 min), while the passive membrane
properties were measured. (B) 11 neurons from control rats
and 9 neurons from epileptic animals were perfused with 0Mg2+ ACSF (30 min). (C) Testing for the effects of JSTX-3
on all studied cells. (D) Washout of JSTX-3 with ACSF
during 10 min and measurement of passive membrane
properties. (E) Perfusion with 0-Mg2+ ACSF. (F) Re-testing
for the effects of JSTX-3 on all studied cells. (G) Perfusion
with normal ACSF. In addition, two cells were studied using
the same protocol, except that cells were treated with
NMDA (25 AM) in normal ACSF instead of being perfused
with 0-Mg2+ ACSF.
3. Results
In order to study the effect of JSTX3 on the
epileptiform activity induced by lowering the concentration of extracellular Mg2+ in control neurons (n = 11),
intracellular recordings were registered on hippocampal
neurons from seven untreated rats kept in normal ACSF
during 10 min (Fig. 1A, stage A) before being perfused
with 0-Mg2+ ACSF for 30 min. Lowering the extracellular concentration of Mg2+ induced spontaneous
interictal and ictal-like discharges in CA1 pyramidal
neurons (Fig. 1A, stage B). Treatment with JSTX-3
(0.1 AM, 5 min) completely blocked the epileptiform
activity induced by 0-Mg2+ ACSF within 50 s (Fig. 1A,
stage C) in a totally reversible way (Fig. 1A, stage D –
G). I V curves carried out before and following the
aforementioned experimental protocol indicate that treatment with 0-Mg2+ ACSF and JSTX-3 does not significantly alter membrane potential (Fig. 1B). As can be seen
in Fig. 1C, lower pannel, JSTX-3 is also able to block
the in vitro epileptiform activity induced by 0-Mg2+
ACSF in hippocampal neurons from rats that had been
rendered epileptic by pilocoparpine injection. Extracellular
recordings obtained in parallel showed that the ictal-like
events registered consisted of paroxysms of repetitive
spike discharges that lasted from 20 to 60 s (Fig. 1C,
upper pannel, stages A to G) which were also blocked by
JSTX-3. I V curves carried out before and after the
aforementioned experimental protocol indicate that treatment of slices taken from pilocarpine-injected animals
with 0-Mg2+ ACSF and JSTX-3 do not significantly alter
membrane potential.
It is known that, on resting conditions, the NMDAr
channel is blocked by Mg2+ ions. Therefore, it is well
possible that the epileptiform activity induced by 0-Mg2+
ACSF perfusion that we observed is due, at least in part, to
overactivation of this subtype of glutamate receptor. To
S.D. Salamoni et al. / Brain Research 1048 (2005) 170 – 176
173
Fig. 1. JSTX-3 blocks the epileptiform activity induced by 0-Mg2+ ACSF in hippocampal neurons from control and pilocarpine-treated rats. (A) Representative
intracellular recording showing the response of a hippocampal pyramidal neuron from a control animal during different experimental stages, as follows: [A]
perfusion with normal ACSF; [B] perfusion with 0-Mg2+ ACSF—note the ictal-like discharges induced by this treatment; [C] application of JSTX-3 (0.1 AM);
[D] washout of JSTX-3 with normal ACSF; [E] perfusion with 0-Mg2+ ACSF; [F] re-application of JSTX-3 (0.1 AM); [G] washout of JSTX-3 with normal
ACSF. Note that JSTX-3 completely blocks the epileptiform activity induced by 0-Mg2+ ACSF in a completely reversible way. (B) Representative curve
showing the IV relationship in a hippocampal pyramidal neuron from a control animal before (h) and after (?) 0-Mg2+ ACSF and JSTX-3 treatments. (C)
Equal to panel (A) but the intracellular recording was taken from a pyramidal neuron of a pilocarpine-treated rat. Note the field potential recording in the upper
part of the figure. (D) Representative curve showing the IV relationship in a hippocampal pyramidal neuron from a pilocarpine-treated animal before (h) and
after (?) 0-Mg2+ ACSF and JSTX-3 treatments.
Fig. 2. JSTX-3 blocks the epileptiform activity induced by iontophoretically applied NMDA in hippocampal neurons. (A) Representative intracellular
recording showing the response of a hippocampal pyramidal neuron from a control animal during different experimental stages, as follows: [A] perfusion with
normal ACSF; [B] iontophoretic application of NMDA (25 AM); [C] application of JSTX-3 (0.1 AM); [D] washout of JSTX-3 with normal ACSF. (B)
Representative photomicrograph showing a pyramidal neuron from the CA1 region of the rat hippocampus filled with biocytin (0.5% w/v).
174
S.D. Salamoni et al. / Brain Research 1048 (2005) 170 – 176
evaluate this hypothesis, we analyzed the effects of JSTX-3
on the ictal-like activity induced in hippocampal neurons by
iontophoretically applied NMDA. Fig. 2 shows a typical
example of the effect of JSTX-3 on the epileptiform
discharges induced by NMDA. The toxin induces an almost
immediate decrease in both the amplitude and frequency of
action potentials together with a sustained membrane
hyperpolarization which is followed by a rapid (within 40
s) blockade of the epileptiform discharges. This blocking
effect was reversed by perfusion with ACSF (10 min). To
confirm that the recording was being performed on hippocampal CA1 neurons, some cells were filled with biocytin as
shown in Fig. 2B.
4. Discussion
In the early 1980s, a new type of neurotoxin was
isolated from the venom of the FJoro_ spider, N. clavata.
This toxin acts on glutamate receptors blocking neurotransmission not only in invertebrates but also in
vertebrates [22]. The initial characterization of Nephila
toxins (JSTX, NSTX) revealed that they contain a variety
of related compounds with a common basic structure,
including a 2,4-dihydroxyphenylacetyl asparaginyl cadaverine moiety connected to a polyamine [15]. Following
this initial characterization, the major component of the
JSTX group of toxins was identified as JSTX-3, which
was later synthesized using different chemical strategies
[15,31].
The main excitatory neurotransmitter l-glutamate acts
on ionotropic (NMDA and non-NMDA) and metabotropic
receptors [12]. The activation of NMDAr increases Na+
and Ca2+ permeability in a voltage-dependent way. NMDA
receptor channel is peculiar because, at resting potential, it
is blocked by Mg2+ [30,34,41]. The lack of magnesium
leads to a state of hyperexcitability due to both the
increase of the NMDAr conductance and the augmentation
of the synaptic release of neurotransmitters [12] and, in
fact, the induction of ictal-like activity elicited by removal
of magnesium from the perfusion medium is an in vitro
tool to evaluate NMDA-mediated epileptic events [3]. Our
study demonstrates that JSTX-3 blocks the epileptiform
activity induced by 0- Mg2+ in hippocampal slices taken
from control and epileptic, pilocarpine-treated rats. In
addition, our experiments also show that JSTX-3 reduces
the ictal-like activity produced by the iontophoretic
application of NMDA.
It is known that binding of spermine and/or spermidine potentiates the conductance through the channel
associated to the NMDAr [19]. Given that JSTX-3 has a
sperminyl-like motif within its structure, it can be
speculated that the blocking effect that this toxin has
on NMDAr can involve the inhibitory interaction with
the spermine binding site. Epileptic phenomena in
chronic experimental animal models, however, have
revealed long-lasting cellular alterations. Most temporal
lobe epilepsy models, including that induced by pilocarpine, have in common the induction of SE, a situation in
which seizures repeat themselves during several hours. It
is believed that prolonged seizures promote the release of
excitatory substances responsible for injuries in sensitive
cerebral structures, such as the hippocampus. As a
consequence of these seizures, there is cellular loss as
well as alterations in the intrinsic properties of nervous
cells and neuronal nets, the latter becoming epileptogenic
[8]. Prolonged stimulation of NMDAr leads to serious
damage, even cellular death, due to calcium toxicity. It
has been proposed that AMPAr also take part in the
process responsible for the epileptiform activity, maybe
promoting changes in the expression of different glutamate receptors subunits such as those that have been
observed in patients with temporal lobe epilepsy [4,6,25]
as well as in many experimental epilepsy models [28].
Saito and coworkers have shown that JSTXs are able to
completely block AMPA and kainate receptor responses in
CA1 pyramidal cells while only partially affecting those
mediated by NMDAr [37]. In addition, in a very
interesting and highly influential set of experiments, Kawai
and coworkers [22] analyzed the effect of the i.c.v.
infusion of JSTX-3 on the behavior of mice and found
that, though the toxin did not produce any abnormal
behavior, it specifically antagonized quisqualate- but not
NMDA or kainate-induced convulsions [23]. Conversely,
our results suggest that the antiepileptic effect of JSTX-3
is, at least partially, mediated by its action on NMDAr. The
reasons for this discrepancy are not clear, but they are
maybe hidden behind the fact that those authors analyzed
the effect of this toxin on kainate-, NMDA- and
quisqualate-mediated responses. Although it was not
known at that time, it is now known that quisqualate has
rather unspecific effects and, in fact, it can activate both
AMPA and metabotropic glutamate receptors [1,21]. When
compared with those previously published, we found that
most published papers emphasize the action of JSTX-3 on
AMPA receptors. Indeed, a number of spider toxins are
effective blocking agents for AMPA-kainate receptor
channels. Glutamate is the neuromuscular transmitter in
insects, and the glutamate receptors of insect muscle are
similar to the AMPA-kainate receptors of vertebrates; the
spiders paralyze their preys by injecting neuromuscular
blocking agents into them [1].
An aspect that must be considered is that most of the
earlier studies were performed using experimental animals
other than the rat such as lobsters, mice and guinea pigs, and
it is now known that the functional and pharmacological
properties of ionotropic glutamate receptor vary greatly
among species [14]. Methodological differences could also
explain why the results are somehow contradictory. The first
studies on JSTXs demonstrated that these toxins can block
glutamate receptors in vivo. The aim of our study was to
characterize its effect in the induction of epileptiform
S.D. Salamoni et al. / Brain Research 1048 (2005) 170 – 176
activity in the CA1 area in vitro [17]. To accomplish that
purpose, we tried to characterize the role played in this
process by NMDA receptors using tools able to facilitate the
functionality of this receptor: the perfusion of a free
magnesium medium and the iontophoretic application of
NMDA. Our results suggest that the antiepileptogenic
action of JSTX-3 is due, at least in part, to the inhibitory
action that this toxin has on the cationic currents evoked
by NMDA receptor activation. In fact, there are other
evidences [33] indicating that JSTXs are not selective for
AMPA receptors, and that, indeed, they can also be
effectively used as NMDAr blocking agents [7,18]. As
has been noticed previously, combined NMDAr/AMPAr
receptor blockade may produce a synergic anticonvulsant
effect.
[10]
[11]
[12]
[13]
[14]
[15]
Acknowledgments
We are grateful to Profs. Matilde Achaval Elena and
Martin Cammarota for their collaboration in preparing this
manuscript. We also thank Miss Fernanda Noal Carlesso her
helpful technical assistance. This study was supported by
CAPES, CNPq, FAPERGS, FAPESP (Bioprospecta Program), PUCRS and Secretaria de C and T-RS. J.C. da Costa
and M.S. Palma are researchers of the National Council of
Scientific and Technological Development (CNPq).
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