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Analysis of Caribbean ciguatoxin-1 effects on frog myelinated axons
and the neuromuscular junction
Ce ´sar Matteia,1, Michel Marquaisa,b, Se ´bastien Schlumbergera, Jordi Molgo ´a,
Jean-Paul Vernouxb, Richard J. Lewisc, Evelyne Benoita,*
aCNRS, Institut de Neurobiologie Alfred Fessard – FRC2118, Laboratoire de Neurobiologie Cellulaire et Mole ´culaire – UPR9040, ba ˆt. 32-33, F-91198
Gif sur Yvette, France
bUnite ´ des Microorganismes d’Inte ´re ˆt Laitier et Alimentaire EA 3213, IFR Icore 146, Universite ´ de Caen, F-14032 Caen Cedex 05, France
cInstitute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia
a r t i c l e i n f o
Received 31 March 2009
Received in revised form 22 July 2009
Accepted 23 July 2009
Available online 29 July 2009
Quantal acetylcholine release
a b s t r a c t
Caribbean ciguatoxin-1 (C-CTX-1) induced, after about 1 h exposure, muscle membrane
depolarisation and repetitive post-synaptic action potentials (APs) in frog neuromuscular
preparations. This depolarising effect was also observed in a Ca2þ-free medium with
a strong enhancement of spontaneous quantal transmitter release, compared with control
conditions. The ciguatoxin-induced increase in release could be accelerated when Ca2þ
was present in the extracellular medium. C-CTX-1 also enhanced nerve-evoked quantal
acetylcholine (ACh) release. At normal neuromuscular junctions loaded with the fluores-
cent dye FM1-43, C-CTX-1 induced swelling of nerve terminals, an effect that was reversed
by hyperosmotic D-mannitol. In myelinated axons, C-CTX-1 increased nodal membrane
excitability, inducing spontaneous and repetitive APs. Also, the toxin enlarged the repo-
larising phase of APs in control and tetraethylammonium-treated axons. Overall, our data
suggest that C-CTX-1 affects nerve excitability and neurotransmitter release at nerve
terminals. We conclude that C-CTX-1-induced up-regulation of Naþchannels and the
inhibition of Kþchannels, at low nanomolar concentrations, produce a variety of functional
dysfunctions that are in part responsible for the human muscle skeletal symptoms
observed in ciguatera. All these dysfunctions seem to result from the subtle balance
between ionic currents, intracellular Naþand Ca2þconcentrations, and engaged second
? 2009 Elsevier Ltd. All rights reserved.
Ciguatoxins (CTX) are responsible for an endemic and
complex human food poisoning, termed ciguatera, which
affects dozen thousands of people each year (Bagnis et al.,
1979; Lewis, 2001). It is due to the consumption of fresh
tropical and subtropical reef fishes that have accumulated,
through the marine food chain, the toxins produced by the
benthic dinoflagellate Gambierdiscus toxicus (Bagnis et al.,
1980; Lewis et al., 2000). This type of poisoning was first
although the term ‘‘ciguatera’’ was created later, in 1866, by
Felipe Poey y Aloy in reference to the name of a venomous
mollusc present in Cuba. Since then, epidemiological
studies showed that ciguatera poisoning has been contin-
uously present in Indo-Pacific and Caribbean areas (Toste-
son et al., 1988; Poli et al., 1997; Pearn, 2001). Ciguatera
poisoning is characterised by a set of gastro-intestinal,
cardiovascular and neurological symptoms (Isbister and
Kiernan, 2005). Pacific ciguatoxins (P-CTX) were reported
to cause primarily neurological symptoms, and Caribbean
* Corresponding author. Tel.: þ33 1 69 82 36 52; fax: þ33 1 69 82 4141.
E-mail address: firstname.lastname@example.org (E. Benoit).
1Present address: De ´le ´gation Ge ´ne ´rale pour l’Armement, Bagneux,
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/toxicon
0041-0101/$ – see front matter ? 2009 Elsevier Ltd. All rights reserved.
Toxicon 56 (2010) 759–767
Author's personal copy
symptoms (Bagnis, 1967; Legrand et al., 1982; Lewis et al.,
1988; Pottier et al., 2001; Achaibar et al., 2007; Wang,
Structural studies have proven that C-CTX molecules are
not completely identical to P-CTX ones, thus defining a new
class of cyclic polyether compounds (Murata et al., 1990;
less toxic to mice than P-CTX. This could be due to structural
differences between the two toxins since, in addition, C-CTX
toxicity, unlike that of P-CTX, is partly destroyed in alkaline
solutions (Vernoux and Lewis,1997).
All the CTX so far described are known to bind to the
receptor-site 5 of voltage-sensitive sodium channels (VSSC)
which, thus, remain permanently open at the resting
membrane potential due to voltage-dependence shift of
their activation to more negative potential values (Benoit
et al., 1986, 1996; Lewis et al., 2000). As a consequence,
nanomolar concentrations of CTX have been reported to
induce spontaneously repetitive action potentials (APs) and
as repetitive endplate potentials that elicited muscle AP in
frog neuromuscular junctions (Molgo ´ et al., 1990; Benoit
et al.,1996; Mattei et al.,1999). The up-regulation of VSSC by
ciguatoxins promotes catecholamine secretion from neuro-
endocrine cells (Mattei et al., 2008). In addition, CTX have
been shown to partially inhibit Kþcurrents, in a voltage-
dependent manner and to increase the cytosolic IP3content
in rat myotubes (Hidalgo et al., 2002).
The aim of the present study was to analyse the effects
of the purified major Caribbean ciguatoxin, Caribbean
ciguatoxin-1 (C-CTX-1), on frog myelinated axons and
neuromuscular junctions. For this purpose, we studied its
electrophysiological action on the two types of prepara-
tions, and visualised the toxin effects on the morphology of
nerve terminals using confocal microscopy.
2. Materials and methods
2.1. Isolated preparations, solutions, drugs and toxins
Experiments werecarried out on both single myelinated
pectoris or sartorius nerve–muscle preparation removed
from male frogs (Rana esculenta) weighing 20–30 g. The
standard Ringer physiological solution used had the
following composition (in mM): NaCl,110.0; KCl, 2.1; CaCl2,
phonic acid (HEPES), 5.0; buffered at pH¼7.25 with NaOH.
In some experiments, Ca2þwas reduced and Mg2þwas
added to the solution as specified in the results. In other
experiments, Ca2þwas substituted by Mg2þ(1.8 mM) and
1 mM ethyleneglycol-bis-(b-amino-ethyl ether) N,N0-tetra-
acetic acid (EGTA) was added to the solution. The following
drugs or toxins were added, when necessary, to solutions
bathing the isolated preparations at final concentrations
indicated in the text: tetrodotoxin (TTX), formamide,
(þ)-tubocurarine and m-conotoxin-GIIIA, purchased from
Sigma (Sigma–Aldrich, Saint Quentin Fallavier, France), and
Merck-Cle ´venot (Nogent sur Marne, France). All salts used
were of analytical grade.
For the extraction and purification of C-CTX-1, eleven
specimens of the pelagic fish Caranx latus (horse eye jack),
often implicated in ciguatera fish poisoning in the Carib-
bean region (Vernoux and Talha, 1989), were collected by
of Saint-Barthelemy in the FrenchWest Indies. The 47 kgof
fish were transported frozen to Caen (France) and stored at
?20?C. Ciguatoxins were extracted by bioassay directed
fractionation, separated and purified to homogeneity by
reverse-phase high performance liquid chromatography
(HPLC) and finally subjected to ion spray mass spectrom-
etry, following procedures described by Lewis et al. (1991)
and modified by Vernoux and Lewis (1997). Dry C-CTX-1
was dissolved in methanol. The solution thus obtained was
divided into several samples, and the methanol was
evaporated under nitrogen to dryness. The pure toxin was
kept dry at ?20?C and diluted immediately before exper-
iments with the standard physiological solution, to give
the final C-CTX-1 concentrations used in the present study.
The chemical structure of C-CTX-1 has been determined
and is depicted in Fig.1. The toxin has a molecular mass of
1141 and is composed of 14 trans-fused, ether-linked rings
with an extremity having a hemiketal moiety (Lewis et al.,
Fig. 1. Chemical structure of the marine polyether toxin C-CTX-1. Molecular mass is 1141.6 Da (Lewis et al., 1998).
C. Mattei et al. / Toxicon 56 (2010) 759–767760
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2.2. Electrophysiological recordings
Single frog myelinated nerves fibres of about 0.5 cm
length were mounted in a five-compartment chamber, as
designed and described previously (Nonner, 1969). The
classical current-clamp technique was used to record APs.
The normal resting potential of fibres was assumed to be
?70 mV, corresponding to 30% inactivation of peak Naþ
current (Sta ¨mplfli and Hille, 1976). The fibre ends were cut
in a solution containing 120 mM KCl, which was used in the
end pools throughout the experiments. In some experi-
ments, Kþchannels were blocked by adding 10 mM TEA
to external solutions. Experiments were performed at
were recorded from isolated neuromuscular preparations
with intracellular glass capillary microelectrodesfilled with
3 M KCl of 4–8 MU resistance, using conventional tech-
niques. Recordings were made either at endplate regions,
visually identified by locating terminal nerve branches
of 1 ms or less, or at extrajunctional areas of the muscle
fibres. In some experiments the motor nerve supplying the
skeletal muscle was drawn into a glass capillary electrode
for stimulation. The nerve was stimulated bysupramaximal
current pulses of 0.05–0.1 ms duration at frequencies of
0.2–0.5 Hz. In some experiments, a dual microelectrode
current-clamp technique, with the aid of an axoclamp-2B
system (Axon Instruments, Fostercity, CA, USA)was used to
trigger and record APs from single muscle fibres. Usually,
the current microelectrode was impaled at about 50 mm
from the recording microelectrode, and the membrane
potential was set at ?100 mV. Electrical signals were after
amplification digitized with the aid of a computerequipped
with an A/D converter (Data Translation 2821, MA, USA) at
a sampling rate of 5–25 kHz. In some experiments, excita-
tion was uncoupled from contraction by treating the
neuromuscular preparations for 21 min with 2 M form-
amide (Del Castillo and Escalona De Motta,1978). After this
treatment, preparations were rinsed for 2–4 h in standard
Ringer solution before recordings.
2.3. Confocal laser scanning microscopy
Living neuromuscular junctions were imaged with
(Molecular Dynamics, California, USA) composed of an
upright Nikon Optiphot-2 microscope equipped with
density transmission filter was used in all experiments. The
aperture setting used was 100 mm. The photomultiplier gain
was kept constant during a given experiment. Neuromus-
cular junctions were routinely visualized with a ?40 water-
immersion objective (0.75 NA). A Silicon Graphics Personal
Iris 4D/35G workstation (Mountain View, CA, USA) and the
software ImageSpace 3.10 (Molecular Dynamics) were used
for control of the scanner module and image analysis. Series
of optical sections were collected using a standard scanning
mode format of 512?512 pixels. From these sections ‘‘look-
through’’ projections were calculated. C-CTX-1 effects on
before and during the action of the toxin. Images from each
experiment were processed identically and stored on
2.4. Staining of living nerve terminals
Nerve terminal membrane structures and perisynaptic
Schwann cell somata of living neuromuscular preparations
were stained with the styryl dye N-(3-triethyl ammo-
niumpropyl)-4-(p-dibutylaminostyryl) pyridinium, dibro-
mide (FM1-43)before imaging.
preparations were exposed for 10–15 min to 2 mM FM1-43,
dissolved in standard Ringer solution, and thereafter rinsed
for about 30 min to remove the free dye. The inability of the
FM1-43 dye to penetrate nerve membranes and the
persistence of the staining, due to its partition only into the
outer membrane leaflet, render this dye particularly useful
for imaging changes in nerve terminal morphology (Betz
et al., 1992).
2.5. Data analysis
Statistical analysis of data was performed by the use of
Student’s-t-test (two tailed). Data were considered signifi-
cant at P<0.05. All values are expressed as the mean-
?SEM of n different preparations.
3.1. Effects on muscle contractile activity
Addition of C-CTX-1 (10–120 nM) to isolated neuro-
muscular preparations bathed in a standard physiological
solution caused, after a short delay (<1 min), visible
spontaneous asynchronous contractions of muscle fibres
that could last for about 2 h. This spontaneous muscle
activity was suppressed either by the addition of 0.5–1 mM
TTX which blocks voltage-dependent Naþchannels in
the nerve and the muscle, or with m-conotoxin GIIIA
(2.5–3.5 mM) whichselectivelyblocksmuscleNaþchannels.
The addition of 5 mM (þ)-tubocurarine to the medium did
not affect the spontaneous contractile activity induced by
C-CTX-1. However, excitation–contraction uncoupling with
2 M formamide (see Materials and methods) completely
prevented the spontaneous contractile activity induced by
C-CTX-1. Since skeletal muscle fibres treated with form-
amide retained their electrical and chemical excitability
properties, most subsequent electrophysiological experi-
ments were performed in neuromuscular preparations pre-
treated with such an uncoupling agent.
3.2. Effects on isolated neuromuscular preparations
As shown in Table 1, C-CTX-1 (40–120 nM) caused, after
a short delay (<1 min) and within 60 min exposure, a dose-
dependent depolarisation of the muscle membrane in
neuromuscular preparations bathed in a standard physio-
logical solution. This membrane depolarisation was pre-
vented by TTX or m-conotoxin GIIIA (not shown). The
blockade of membrane depolarisation, induced by pre-
treating the preparations with TTX before adding C-CTX-1
C. Mattei et al. / Toxicon 56 (2010) 759–767761
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(60 nM), was suppressed by washing preparations with
a normal Ringer solution. This indicates that C-CTX-1 was
tightly bound to its receptor and virtually not removable.
C-CTX-1 (100 nM), added to the Ringer solution,
produced a decrease of muscle AP amplitude when the
muscle was directly stimulated (data not shown). Under
this condition, the excitability threshold was decreased. In
addition, C-CTX-1 (100 nM) induced spontaneous and
repetitive APs (Fig. 2A). These C-CTX-1 effects, which were
not observed in a Ca2þ-free solution added with the
calcium chelator EGTA even at a high toxin concentration
(440 nM), were likely due to a pre-synaptic action. Indeed,
C-CTX-1 was observed to induce spontaneous and repeti-
tive endplate potentials (EPPs), at frequencies ranging
between 7 and 37 Hz, either in the absence of stimulation
or in response to single nerve stimulation (Fig. 2B and C).
These EPPs, when they had sufficient amplitude to reach
the threshold for AP generation, triggered trains of APs (see
Fig. 2A). These APs werecompletelysuppressed byblocking
both nerve and muscle Naþchannels with TTX, but EPPs
were not abolished by blocking only muscle Naþchannels
with m-conotoxin GIIIA.
As shown in Fig. 3, in the presence of C-CTX-1 (100 nM),
beginning of sequences of synaptic events and then stabi-
lized at about 16 ms (Fig. 3A), with a normal distribution
(Fig. 3B). This indicates that the frequency of EPPs was high
at the beginning of sequences and then stabilized at a lower
level of about 50–60 Hz. Spontaneous multiquantal EPPs
were facilitated or depressed according to the presence of
a silent period between sustained synaptic activities. In
a Ringer solution containing low Ca2þ(1 mM), high Mg2þ
(10 mM) and neostigmine (3 mM), C-CTX-1 (120 nM)
decreased, as a function of time, the amplitude of evoked
EPP, followed by a complete blockade of responses 80 min
after toxin addition (Fig. 4). However, a sudden and tran-
sient increase in evoked EPP amplitude was observed
50 min after C-CTX-1 application (Fig. 4). This increase was
concomitant to C-CTX-1-induced trains of repetitive EPPs
inset of Fig. 4). These results, which are representative of 4
additional experiments, indicate thatthe quantal contentof
evoked EPPs decreased with time, leading to a complete
blockade of acetylcholine (ACh) synchronous release. It is
likely that this blockade is not due to the exhaustion of
neurotransmitter stocks, since high-rate MEPPs could still
be recorded from the same neuromuscular junction (see
Effect of C-CTX-1 on the resting membrane potential of skeletal muscle
fibres bathed in a standard physiological solution.
C-CTX-1 (nM) Resting membrane
Fig. 2. Spontaneous and repetitive activity recorded in neuromuscular preparation treated with C-CTX-1 (100 nM). (A) Spontaneous and repetitive APs recorded
in a junctional region of the muscle fibre after addition of C-CTX-1 to the standard physiological solution. Note the curvature at the foot of APs, showing that they
were triggered by an EPP. The last trace shows an EPP that failed to elicit an AP. (B and C) Spontaneous multiquantal EPPs recorded from another junction, 1 min
(B) and 5 min (C) after addition of C-CTX-1 to the solution. Note the increase in MEPP frequency and spontaneous multiquantal EPPs. (D) Failure of nerve
stimulation to elicit an EPP 80 min after C-CTX-1 action. Note that MEPPs can still be observed. (A–D) Neuromuscular preparation pre-treated with formamide to
uncouple excitation from contraction. Vertical scale bars: 20 mV (A), 2 mV (B) and 0.5 mV (C and D).
C. Mattei et al. / Toxicon 56 (2010) 759–767 762
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Fig. 2D), but result from the reduction of the pre-synaptic
action potential due to nerve terminal depolarization.
The addition of C-CTX-1 (120 nM) to a standard Ringer
solution produced a time-dependent enhancement of
spontaneous quantal ACh release, detected as an increased
MEPP frequency. Plotted in semi-logarithmic scale, the
time-course of this increase could be well described by
a sigmoid which attained a maximum value of about
400 Hz, 80 min after toxin addition (Fig. 5A). The C-CTX-1-
induced increase in MEPP frequency was dose-dependent
(between 12 and 120 nM of toxin),and occurred even in the
absence of external Ca2þ, although at a lower rate as
compared to Ca2þbeing present in the external medium
To visualize the morphological changes induced by
C-CTX-1 to motor nerve terminals, during the increased
quantal transmitter release, we stained frog neuromuscular
with the toxin in a Ca2þfree medium containing EGTA. As
seen in Fig. 6, C-CTX-1 (120 nM) significantly increased, as
a function of time, the projected area of nerve terminals.
This swelling could be partially reversed by adding
D-mannitol to the external medium, which increased the
osmolarityof the external solution. Thisresult suggests that
the toxin induces, together with an entry of Naþions,
a water influx into nerve terminals.
3.3. Effects on single myelinated nerve fibres
External application of C-CTX-1 (1–120 nM) to single
myelinated nerve fibres produced mainly an increase in the
nodal membrane excitability, as revealed by AP studies.
Thus, under control conditions, a brief depolarising stim-
ulus evoked a single AP in motor or sensory nerve fibres
(Fig. 8A, left trace). However, during a long-lasting depo-
larising stimulus, motor and sensory nerve fibres generate
single and repetitive APs, respectively (see, for example,
Fig. 7). This has been attributed, at least in part, to differ-
ences in the threshold potential between sensory and
motor nerve fibres (Neumcke, 1981). C-CTX-1 caused
a slight depolarisation of the membrane of about 2.5 and
6.5–13 mV with 1 and 10–120 nM of toxin, respectively. As
a consequence, spontaneous and repetitive APs were
usually observed in the presence of 10–120 nM C-CTX-1
(Fig. 8A, right trace). Although spontaneous APs were not
detected in the presence of 1 nM of toxin, the slight C-CTX-
1-induced membrane depolarisation was sufficient to
Fig. 3. Distribution of intervals between spontaneous repetitive EPPs
recorded from a single neuromuscular junction in the presence of C-CTX-1
(100 nM) as a function of the sequence number of EPPs (A), and as a function
of the intervals between synchronous releases (B).
Fig. 4. Time-course of evoked EPP amplitude in the presence of C-CTX-1 (120 nM). All data were recorded from the same neuromuscular junction bathed in
a Ringer solution containing 1 mM Ca2þ– 10 mM Mg2þand neostigmine (3 mM). The preparation was pre-treated with formamide to uncouple excitation from
contraction. Inset: facilitation of sequential spontaneous EPPs recorded 60 min after C-CTX-1 addition; scale bars: 4 ms (horizontal) and 0.4 mV (vertical).
C. Mattei et al. / Toxicon 56 (2010) 759–767763
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decrease the threshold potential of nerve fibres by about
20%. As a result, repetitive APs were observed when long-
lasting depolarising stimuli were applied to preparations.
Thus, a motor nerve fibre, as defined under control condi-
tions, responded like a sensory one in the presence of 1 nM
C-CTX-1 (Fig. 7). Therefore,1 nM C-CTX-1 seemed to be the
‘‘threshold’’ concentration for increasing the excitability of
the nodal membrane of single myelinated nerve fibres.
The spontaneous and repetitive APs appeared about
8 min and 5–30 s after the application of 10 and 50–120 nM
(Fig. 8A, right trace) and was not dependent on the toxin
concentration (between 10 and 120 nM). However, after
a few minutes, spontaneous AP discharges appeared as
bursts interrupted by silent periods (Fig. 8B, right trace).
These periodswere of longerdurationandmorefrequentas
the time of toxin application increased. The enhanced
excitability, giving rise to spontaneous and repetitive APs
caused by low concentrations of C-CTX-1 (<50 nM) was
reversed after 45 min of washing nerve fibres with a toxin-
free standard solution, provided the toxin was applied for
extensive washing for up to 75 min, which indicates that
C-CTX-1 was very tightly bound to its receptor.
In the presence of 10–120 nM C-CTX-1, the amplitude of
spontaneous and repetitive APs was decreased by 22 ?2%
repolarisation, increased to 164?12% (n¼5) of control
value (see Fig. 8A and B, left traces). These modifications
AP duration was further increased to 476? 82% (n¼4) of
control value bysubsequent addition of TEA (10 mM) to the
external solutions containing 10–120 nM C-CTX-1 (Fig. 8C,
left trace). Under control conditions, the addition of 10 mM
TEAtothe standard Ringer solutionproduced an increase to
230? 60% (n¼4) in the AP duration (Fig. 8D, left trace). The
subsequent addition of 100 nM C-CTX-1 to the external
solution containing TEA further increased the AP duration
to about 210% (Fig. 8D, middle trace), giving an increase to
about 440% of control value, which is close to the value
obtained when C-CTX-1 was added before TEA, i.e. about
480% (see above). It is worth noting that, in the presence of
C-CTX-1 and TEA, spontaneous activities could still be
observed (Fig. 8C and D, right traces) although the
frequencyof repetitive APs, about 5 Hz, was lower than that
previously reported in the absence of TEA, i.e. about 50 Hz
(see Fig. 8A and B, right traces).
In contrast to Pacific ciguatoxins which have been
aspects, less is known regarding Caribbean ciguatoxins.
However,theirrespective structuresareclosely related,and
both of them seem to induce the same kind of symptoms,
even if neurological ones are predominantly observed
potent activators of VSSC: they bind, like brevetoxins, to
receptor-site 5 of channels at nanomolar concentrations,
which induces both a shift of their activation towards more
negative membrane potentials and a partial inhibition of
their inactivation (Lombet et al., 1987; Benoit et al., 1996).
However, recent studies suggest that ciguatoxins and bre-
vetoxins could also bind to other ion channels such as Kv
channels at low concentrations (Hidalgo et al., 2002) and
TRPV1 channels at higher concentrations (Cuypers et al.,
2007). These recent data throw light on the possible
versatility of polyether molecules to interact with different
kind of channel receptors.
Our investigation provides evidence that C-CTX-1
increases asynchronous muscle contractions, nerve excit-
ability and both spontaneous and evoked release of quantal
ACh release from motor nerve terminals. These neuro-
musculareffectswere abolished byTTX, providing
0 1240 60 801200 120
Fig. 5. Effects of C-CTX-1 on spontaneous quantal ACh release. (A) Time-course of the increase in MEPP frequency produced by the addition of C-CTX-1 (120 nM)
to the standard Ringer solution. All data were obtained from the same neuromuscular junction pre-treated with formamide to uncouple excitation from
contraction. Each point corresponds to the mean ?SEM of 10 measurements executed in a 10 ms time period. Inset: MEPPs recorded after C-CTX-1 addition; scale
bars: 100 ms (horizontal) and 0.4 mV (vertical). (B) Effects of C-CTX-1 concentration on MEPP frequency recorded after 1 h in Ringer solution in the absence, or in
the presence of external Ca2þ(1.8 mM). Each column represents the mean ?SEM of data obtained in 3–8 different preparations.
C. Mattei et al. / Toxicon 56 (2010) 759–767764
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evidence that C-CTX-1 acts through an activation of VSSC.
Our data clearly show that C-CTX-1, at nanomolar
concentrations, targets both pre- and post-synaptically the
skeletal neuromuscular junction. It is worth noting that,
although the affinity constant of the toxin cannot be
accurately determined under our experimental conditions,
it is likely that it does not markedly differ for pre- and
post-synaptic effects since these effects occurred in the
same concentration range of C-CTX-1, i.e. 10–120 nM.
Post-synaptically, C-CTX-1 produces TTX- or m-conotoxin
Fig. 6. Effects of C-CTX-1 (120 nM) on the morphology of motor nerve terminals in situ. Images represent reconstitutions of look-through projection of optical
sections. Nerve terminals were stained with FM1-43 and observed before (A) and 75 (B), 150 (C) min after addition of C-CTX-1B (nM) in a Ca2þ-free medium
containing EGTA (2 mM). Note the time-dependent swelling of living structures, including nerve terminals branches and perisynaptic Schwann cell soma (SCS).
(D) Same neuromuscular junction imaged 20 min after addition of D-mannitol (100 mM) to the C-CTX-1-containing solution. The distribution of the membrane
dye FM1-43 changes over time, due to the movements of vesicles during endo–exocytosis.
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junction clearly show that C-CTX-1 increases both sponta-
neous and Ca2þ-dependent evoked quantal ACh release,
recorded respectively as MEPPs and EPPs. The latter trigger
repetitive post-synaptic APs which set off spontaneous and
repetitive muscle contractions post-synaptically. In addi-
tion, nerve terminals were morphologically affected by C-
CTX-1: we observed a significant swelling of nerve
branches, suggesting a ciguatoxin-induced water influx, in
response to the entry of Naþions into nerve terminals. This
swelling could be reversed by adding D-mannitol to the
external medium, making it one of the currently used
treatments to ciguatera (Palafox et al., 1988). All these
physiological effects, which were also previously described
using P-CTX-1B, P-CTX-4B and PbTx-3, are typical to those of
toxins activating VSSCs (Molgo ´ et al., 1990; Meunier et al.,
1997; Mattei et al.,1997).
In frog myelinated axons, C-CTX-1 (1–120 nM) was
reported to depolarise the membrane, and to induce
spontaneous and repetitive APs with increased duration.
These effects are the results of the binding of the toxin to
both VSSC and voltage-gated Kþchannels (Bidard et al.,
1984; Hidalgo et al., 2002). By targeting VSSC, C-CTX-1
shifts their activation to more negative potentials, thus
depolarizing the nerve fibre membrane. This explains the
Fig. 7. Actionpotentials evoked bya long-lasting (95 ms) depolarising stimulus and recorded from a single myelinated nerve fibre, before (A) and after (B) C-CTX-1
(1 nM) addition to the bath solution. Note the repetitive APs after the addition of the toxin.
Fig. 8. Effects of C-CTX-1 (100 nM) on AP recorded from single isolated myelinated nerve fibres. (A–C) Control AP evoked by a brief (0.5 ms) depolarising stimulus
(A, left trace), spontaneous APs recorded without any stimulus 5 min (A, right trace) and 10 min (B) after C-CTX-1 addition, and 10 min after subsequent addition
of 10 mM TEA to the C-CTX-1-containing solution (C). (D) AP evoked by a brief (0.5 ms) depolarising stimulus and recorded in the presence of 10 mM TEA (left
trace), and spontaneous APs recorded without any stimulus 10 min after C-CTX-1 addition to the TEA-containing solution (middle and right traces).
C. Mattei et al. / Toxicon 56 (2010) 759–767 766
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observed repetitive APs (Benoit et al., 1996; Mattei et al.,
1999). The inhibitory effect of C-CTX-1 on Kþchannels
results in an increased AP duration. The subsequent addi-
tion of the Kþchannel inhibitor TEA contributes to prolong
APs, and finally to decrease the frequency of ciguatoxin-
induced repetitive APs. The double mode of action of
C-CTX-1 ondifferentchannelsproduces a complexeffecton
myelinated nerve fibres.
Our functional data illustrate the versatility of a cigua-
toxin on one of its physiological targets, the motor nerve
fibres. A combination of electrophysiological recordings
and morphological analysis on nodes of Ranvier, nerve
terminals and muscle shows that the ciguatoxin-induced
up-regulation of VSSC and the inhibition of Kþchannels, at
nanomolar concentrations, produce a variety of functional
dysfunctions that are in part responsible for the human
muscle skeletal symptoms observed in ciguatera (Pearn,
2001; Achaibar et al., 2007).
This work was supported in part by grant STC-CP2008-
1-555612-Atlantox (to E.B. and J.M.).
Conflicts of interest
The authors declare that there are no conflicts of
Achaibar, K.C., Moore, S., Bain, P.G., 2007. Ciguatera poisoning. Pract.
Neurol. 7, 316–322.
Bagnis, R., Chanteau, S., Chungue, E., Hurtel, J.M., Yasumoto, T., Inoue, A.,
1980. Origins of ciguatera fish poisoning: a new dinoflagellate,
Gambierdiscus toxicus Adachi and Fukuyo, definitively involved as
a causal agent. Toxicon 18, 199–208.
Bagnis, R., 1967. Clinical aspects of ciguatera fish poisoning in French
Polynesia. Hawaii Med. J. 28, 25.
Bagnis, R., Kuberski, T., Laugier, S., 1979. Clinical observations on 3009
cases of ciguatera (fish poisoning) in the South Pacific. Am. J. Trop.
Med. Hyg. 28, 1067–1073.
Benoit, E., Juzans, P., Legrand, A.M., Molgo ´, J., 1996. Nodal swelling
produced by ciguatoxin-induced selective activation of sodium
channel in myelinated nerve fibres. Neuroscience 71, 1121–1131.
Benoit, E., Legrand, A.M.,Dubois, J.M.,1986.Effects of ciguatoxin oncurrent
Betz, W.J., Mao, F., Bewick, G.S., 1992. Activity-dependent fluorescent
staining and destaining of living vertebrate motor nerve terminals.
J. Neurosci. 12, 363–375.
Bidard, J.N., Vijverberg, H.P.M., Frelin, C., Chungue, E., Legrand, A.M.,
Bagnis, R., Lazdunski, M., 1984. Ciguatoxin is a novel type of Naþ
channel toxin. J. Biol. Chem. 259, 8353–8357.
Cuypers, E., Yanagihara, A., Rainier, J.D., Tytgat, J., 2007. TRPV1 as a key
determinant in ciguatera and neurotoxic shellfish poisoning. Bio-
chem. Biophys. Res. Commun. 361, 214–217.
Del Castillo, J., Escalona De Motta, G., 1978. A new method for excitation-
Isbister, G.K., Kiernan, M.C., 2005. Neurotoxic marine poisoning. Lancet
Neurol. 4, 219–228.
Legrand, A.M., Galonnier, M., Bagnis, R., 1982. Studies on the mode of
action of ciguateric toxins. Toxicon 20, 311–315.
the human response to ciguatera in Australia. In: Proceedings of the
Sixth International Coral Reef Symposium, Townsville, vol. 3 67–72.
Lewis, R.J., Sellin, M., Poli, M.A., Norton, R.S., Macleod, J.K., Sheil, M.M.,
1991. Purification and characterization of ciguatoxins from moray eel
(Lycodontis javanicus Muraenidae). Toxicon 29, 1115–1127.
Lewis, R.J., Vernoux, J.P., Brereton, M., 1998. Structure of Caribbean cigua-
toxin isolated from Caranx latus. J. Am. Chem. Soc.120, 5914–5920.
Lewis, R.J., Jones, A., Vernoux, J.P., 1999. HPLC/tandem electrospray mass
spectroscopy for the determination of sub-ppb levels of Pacific and
Caribbean ciguatoxins in crude extracts of fish. Anal. Chem. 71,
Lewis, R.J., Molgo ´, J., Adams, D.J., 2000. Ciguatera toxins: pharmacology of
(Ed.), Seafood and Freshwater Toxins. Pharmacology, Physiology and
Detection. Marcel Dekker, Inc., New-York, pp. 419–447 (Chapter 20).
Lewis, R.J., 2001. The changing face of ciguatera. Toxicon 39, 97–106.
Lombet, A., Bidard, J.N., Lazdunski, M., 1987. Ciguatoxin and brevetoxins
share a common receptor site on the neuronal voltage-dependent
Naþchannel. FEBS Lett. 219, 355–359.
Mattei, C., Benoit, E., Juzans, P., Legrand, A.M., Molgo ´, J., 1997. Gambier-
toxin (CTX-4B), purified from wild Gambierdiscus toxicus dinoflagel-
lates, induces Naþ-dependent swelling of single frog myelinated
axons and motor nerve terminals in situ. Neurosci. Lett. 234, 75–78.
Mattei, C., Molgo ´, J., Marquais, M., Vernoux, J.P., Benoit, E., 1999. Hyper-
osmolar D-mannitol reverses the increased membrane excitability
and the nodal swelling caused by Caribbean ciguatoxin-1 in single
frog myelinated axons. Brain Res. 847, 50–58.
Mattei,C., Wen, P.J., Nguyen-Huu,
Bourdelais, A.J., Lewis, R.J., Baden, D.G., Molgo ´, J., Meunier, F.A., 2008.
Brevenal inhibits Pacific ciguatoxin-1B-induced neurosecretion from
bovine chromaffin cells. PLoS One 3 (10), e3448. doi:10.1371/journal.
Meunier, F.A., Colasante, C., Molgo ´, J.,1997. Sodium-dependent increase in
quantal secretion induced by brevetoxin-3 in Ca2þ-free medium is
associated with depletion of synaptic vesicles and swelling of motor
nerve terminals in situ. Neuroscience 78, 883–893.
Molgo ´, J., Comella, J.X., Legrand, A.M., 1990. Ciguatoxin enhances quantal
transmitter release from frog motor nerve terminals. Br. J. Pharmacol.
Murata, M., Legrand, A.M., Ishibashi, Y., Fukui, M., Yasumoto, T., 1990.
Structure and configurations of ciguatoxin from the moray eel Gym-
nothorax javanicus and its likely precursor from the dinoflagellate
Gambierdiscus toxicus. J. Am. Chem. Soc. 112, 4380–4386.
Neumcke, B.,1981. Differences in electrophysiological properties of motor
and sensory nerve fibres. J. Physiol. Paris 77, 1135–1138.
Nonner, W., 1969. A new voltage clamp method for Ranvier nodes.
Pflu ¨gers Arch. 309, 176–192.
Palafox, N.A., Jain, L.G., Pinano, A.Z., Gulick, T.M., Williams, R.K., Schatz, I.J.,
1988. Successful treatment of ciguatera fish poisoning with intrave-
nous mannitol. J. Am. Med. Assoc. 259, 2740–2742.
Pearn, J., 2001. Neurology of ciguatera. J. Neurol. Neurosurg. Psychiatr. 70,
Poli, M., Lewis, R.J., Dickey, R., Musser, R., Buckner, C., Carpenter, L., 1997.
Identification of Caribbean ciguatoxins as the cause of an outbreak of
fish poisoning among U.S. soldiers in Haiti. Toxicon 35, 733–741.
Pottier, I., Vernoux, J.P., Lewis, R.J., 2001. Ciguatera fish poisoning in the
Caribbean islands and Western Atlantic. Rev. Environ. Contam.
Toxicol. 168, 99–141.
Sta ¨mplfli, R., Hille, B., 1976. Electrophysiology of the peripheral myelin-
ated nerve. In: Llina `s, R., Precht, W. (Eds.), Frog Neurobiology.
Springer, Berlin, Heidelberg, New-York, pp. 3–32.
Tosteson, T.R., Ballantine, D.L., Durst, H.D., 1988. Seasonal frequency of
ciguatoxic barracudain southwest
Vernoux, J.P., Lewis, R.J., 1997. Isolation and characterisation of Caribbean
ciguatoxins from the horse-eye Jack (Caranx latus).Toxicon 35, 889–900.
Vernoux, J.P., Talha, F., 1989. Fractionation and purification of some
muscular and visceral ciguatoxins extracted from Caribbean fish.
Comp. Biochem. Physiol. 94B, 499–504.
Wang, D.Z., 2008. Neurotoxins from marine dinoflagellates: a brief
review. Mar. Drugs 6, 349–371.
T.D., Alvarez,M., Benoit,E.,
C. Mattei et al. / Toxicon 56 (2010) 759–767767