Inhibitory effects of tutin on glycine receptors in spinal neurons
Jorge Fuentealbaa,c,⁎, Leonardo Guzmána,c, Paula Manríquez-Navarroa, Claudia Pérezb,c,
Mario Silvab, José Becerrab,c, Luis G. Aguayoa,c
aDepartment of Physiology, University of Concepcion, Chile
bDepartment of Botany, University of Concepcion, Chile
cPatagonian Ecosystems Research Center (CIEP), Chile
Received 21 June 2006; received in revised form 7 December 2006; accepted 14 December 2006
Available online 17 January 2007
We studied the effects of tutin, a sesquiterpenoid obtained from Coriaria ruscifolia subspecie ruscifolia, a native poisonous Chilean plant, on
spinal glycine receptors using patch clamp recordings. In addition, cytosolic Ca2+transients and activation of cAMP response element-binding
protein (CREB) were measured in the presence of tutin. Application of tutin (1–1000 μM) inhibited the glycinergic evoked current in a
concentration-dependent manner. Moreover, the frequency of spontaneous Ca2+spikes and spontaneous synaptic activity (AMPAergic events) was
augmented and correlated with an increase in phosphorylated CREB levels, suggesting an enhancement in neuronal excitability. These results may
explain the toxic effects of the plant characterized by seizures and convulsions with subsequent coma and death seen in humans and mice.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Spinal neuron; Tutin; Picrotoxin; Synaptic transmission; Strychnine; Coriaria ruscifolia
Extracts from Coriariaceae have been used in Chinese
traditional medicine to treat mental disease; however, in some
cases seizures were reported. In addition, it was recently
described that an extract of Coriaria lactone induced strong
seizures in rats (Wang et al., 2003). Therefore, this study
suggests that the mechanism of action of this extract may be
central nervous system (CNS). Coriaria ruscifolia subspecie
1982). It was previously suggested that the toxic effect of
Coriariaceae may be produced by tutin and coriamyrtin, two of
its main components (Reyes et al., 1998; Wang et al., 2003).
to picrotoxin (Kudo et al., 1984). Therefore, the toxic effects of
C. ruscifolia (Garcia Martin et al., 1983) might be on GABAA
and glycine receptors (Takeuchi and Takeuchi, 1969). Using
extracellular recordings, it was reported that tutin, in the
micromolar range, inhibited GABA induced depolarizations in
the frog spinal cord (Kudo et al., 1984) suggesting the
involvement of GABAAreceptors. It is presently unknown if
tutin can interfere with glycine receptors, which are expressed in
lower regions of the CNS (Lynch, 2004).
Glycine receptors are pentameric structures composed of α
(1–4) and β subunits. Activation of these receptors by glycine
induces a rapid increase in Cl−conductance which is
accompanied by a reduction in the excitability of neurons
involved in pain, motor coordination and respiratory rhythms
(Aguayo et al., 2004; Lynch, 2004). These receptors might also
be implicated in seizure generation, since potentiation of
glycinergic transmission was shown to block seizures (Seiler
and Sarhan, 1984). Therefore, we examined the effect of tutin
on spinal glycine receptors in an intent to explain the toxicity of
the plant described in mice and humans (Garcia Martin et al.,
1983; Wang et al., 2003).
European Journal of Pharmacology 559 (2007) 61–64
⁎Corresponding author. Laboratory of Neurophysiology, Department of
Physiology, University of Concepcion, PO Box 160-C, Concepción, Chile. Tel.:
+56 41 207318; fax: +56 41 245975.
E-mail address: email@example.com (J. Fuentealba).
0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
2. Materials and methods
The animals were manipulated according to ethical proce-
dures for animal management as outlined by the Animal Use
and Care Committee at the University of Concepcion.
2.1. Tutin isolation
Tutin was isolated from dried leaves of C. ruscifolia locally
collected. The first extraction was made in methanol and then
fractioned with solvents of incremental polarity (from hexane to
ethyl acetate). The final purification was made from chlor-
oformic and ethyl acetate portions using SiO2 column
chromatography and crystallization. The purity (N99%) was
assayed with1H-RMN and13C-RMN as previously reported
(Okuda et al., 1987; Perry et al., 2001).
2.2. Cell culture
Mouse (C57BL/J6) spinal cord neurons obtained from five to
six embryos (13–14 days) were plated at 300,000 cells/ml into
35-mm tissue culture dishes coated with poly-L-lysine (MW
350 kDa, Sigma Chemical, St. Louis, MO). The neuronal
feeding medium consisted of 90% minimal essential medium
(GIBCO, Grand Island, NY), 5% heat-inactivated horse serum
(GIBCO), 5% fetal bovine serum (GIBCO) and a mixture of
nutrient supplements (Aguayo and Pancetti, 1994). Fresh media
was replaced every 3 days. Experiments were performed on 14
days in vitro neurons.
2.3. Intraperitoneal tutin administration
In vivo experiments were carried out on Wistar male rats
(250 g, n=4). The small number of animals used for in vivo
toxicity studies was institutionally justified to reduce excessive
suffering according to the triple R principle. Tutin diluted in
Ringer's solution to concentrations of 1, 3, 5, and 8 mg/kg at a
final volume of 1 ml, was intraperitoneally injected beginning
with the lower dose. The time required to elicit toxic responses
was evaluated according to the following parameters: 1)
immobility: cessation of exploratory capacity, 2) muscle
spasm: induction of muscle tremors, 3) seizures: a general
convulsive crisis and 4) death: cardio-respiratory collapse.
2.4. Electrophysiological recordings
Voltage-clamp recordings were performed in whole cell
configuration and acquired with an Axon 200-B amplifier
(Axon Instruments, Union City, CA). Patch-clamp microelec-
trodes were filled with (in mM): 140 KCl, 10 BAPTA, 10
HEPES (pH 7.4), 4 MgCl2, 0.3 GTP and 2 ATP-Na2,
300 mOSM. The external solution contained (in mM): 150
NaCl, 5.4 KCl, 2.0 CaCl2, 1.0 MgCl2, 10 HEPES (pH 7.4) and
10 glucose. The holding potential was fixed at −60 mV,
recordings were filtered at 5 kHz with a low pass Bessel filter.
The solutions were prepared fresh every day. The recordings
were made by applying short (2 s) pulses of glycine (30 μM)
every 1 min with a rapid perfusion system. After stabilizing the
glycinergic current amplitude, tutin was co-applied and the
amplitude of the current to glycine re-evaluated. We applied the
full range of tutin concentrations (1–1000 μM) to each neuron
in order to obtain a concentration-response curve.
Spontaneous synaptic activity was classified according to the
decay time constant (τ) of single synaptic events as follows:
AMPAergic τb10 ms, glycinergic τ∼10–40 ms, and GABAer-
gic τN40 ms (van Zundert et al., 2004). Decay time constant,
amplitude, frequency and number of events were analyzed with
the Minianalysis program (Synaptosoft Inc).
Spontaneous Ca2+transients were detected with Calcium
Green® (Molecular Probes). Neurons were incubated with
Fig. 1. Effects of tutin on in vivo toxicity and evoked glycine current. A: Toxic
effects of tutin observed at different doses and times. The inset shows the
structure of tutin. B: Current traces evoked by applications of glycine (30 μM)
alone and with tutin (200 μM). C: The graph shows the effect of tutin at different
concentrations of glycine (10–100 μM). Pulses of glycine were applied for 2 s
every 1 min. Tutin was co-applied with glycine. The data shows the normalized
current as percentage of control (n=5).
62J. Fuentealba et al. / European Journal of Pharmacology 559 (2007) 61–64
3μM Calcium Green during30 min and after extensive washing
they were placed in a perfusion chamber on a microscope
(Nikon, TE 2000). Changes in the cytosolic Ca2+was acquired
with a CCD camera (12BIT, SensiCam, PCO) and Lambda 10–
2 (Sutter Instruments, USA) interface. Each image was recorded
every 1 s and time exposition was 200 ms.
2.6. cAMP response element-binding protein analysis
For cAMP response element-binding protein (CREB)
measurements, neurons were treated with different concentra-
tions of tutin for 60 min. The cells were then lysed and the
protein concentration of the extracts was measured. Equal
amounts of protein (6 μg) were analyzed by polyacrylamide gel
electrophoresis (PAGE). The proteins were transferred to
nitrocellulose and Ser133 phosphorylated CREB (CREB-P)
was identified by Western blot using a specific antibody
(Upstate™). The signal of CREB-P was compared to the signal
of a load control (Gβ protein) whose expression level was the
same in all conditions using the ImageJ software.
2.7. Data analysis
Data are expressed as means±SEM. Statistical comparisons
were performed using Student's t-test. pb0.05 was considered
3.1. Animal toxicity
The toxicity of purified tutin (inset Fig. 1A) was examined in
when the lower dose was used (1 mg/kg). As the dose was
incremented, the animals showed progressive symptoms of im-
to induce toxicity decreased with increased dosage (n=4).
3.2. Effects on whole-cell currents
Short applications of glycine (30 μM) to spinal neurons
elicited a Cl−current that was stable in control condition for at
et al., 2004). The amplitude of the current was reduced by
approximately 50% when tutin (200 μM) was co-applied with
glycine (Fig. 1B). This current inhibition was maintained and did
not show further increments with repetitive applications. In
addition, the inhibition induced by tutin in spinal neurons was
only slightly reversible (20±11%) after 3 min of washout with
external solution (n=7). The effect of tutin was concentration
dependent, thus it was reduced by 22±8% with 100 μM and 44±
11% with 200 μM (n=7). In addition, we found that the effect of
tutin was not reversed even using a supramaximal concentration
of glycine (100–500 μM, 57±7% of control), suggesting a
noncompetitive mechanism (Fig. 1C), similar to picrotoxin
(Lynch, 2004). Interestingly, the effect of tutin (200 μM) was
not selective for glycine receptors, since we found that it also
of control in the same neurons. On the other hand, the inward
current induced by application of glutamate was not significantly
altered by tutin (82±7% of control, n=5).
3.3. Effects on synaptic transmission and calcium transients
glycine receptors and GABAergic activities, this should be
Fig. 2. Effects of tutin on Ca2+transients and phosphorylated-CREB.
A: Fluorometric recordings of spontaneous calcium activity in a single neuron.
The neurons were incubated with 3 μM Calcium Green® and the images were
acquired in the absence and presence of tutin (200 μM) perfused into the bath.
The increments are measured over basal fluorescence and expressed as Arbitrary
Units of Fluorescence (AUF; n=5). B: Frequency (Hz) of spontaneous Ca2+
transients before (bar with horizontal lines) and during the perfusion of tutin
(white bar; n=14;⁎⁎pb0.01). C: Original traces of spontaneous synaptic
activity from control and tutin (200 μM) conditions. Note that the evoked
current induced by 30 μM glycine was also reduced. D: Western blot analysis of
lysates from cells treated with tutin (100–500 μM) and detected with an
antibody against phosphorylated-CREB (ser 133) (CREB-P). The position of
CREB-P is indicated by the arrowhead (n=3).
63J. Fuentealba et al. / European Journal of Pharmacology 559 (2007) 61–64
synaptic activity. Fig. 2A shows that neurons responded with an
increase in spontaneous Ca2+transients upon application of tutin
(200 μM). Fig. 2B shows that the frequency increased from
0.016±0.005 Hz in control condition to 0.040±0.007 Hz in
the presence of tutin (n=14;⁎⁎pb0.01). These results confirm
that tutin augmented overall neuronal network activity. Addition-
ally, with electrophysiological experiments, we found that the
sustained perfusion of tutin (200 μM) into the bath caused an
increment of 53±14% in overall spontaneous synaptic activity
(n=6, pN0.05). Fig. 2C shows the increment of synaptic trans-
to an increase in the number of AMPAergic currents compared to
that before its application (20±7 vs 47±10%, n=6,⁎pb0.05).
3.4. cAMP response element-binding protein phosphorylation
The marked increase in neuron activity observed in the
presence of tutin might be associated to an increase in Ca2+
dependent signal transductions,since itisknownthat changes in
the concentration of this cation affect Ca2+-dependent transcrip-
tion factors (Dolmetsch et al., 1997, 1998). Therefore, we
decided to examine the activation of CREB, a key marker for
neuronal activity, which is known to be up regulated during
epileptogenic seizure induction and myoclonic status (Dash
et al., 1991; Josselyn and Nguyen, 2005). Neurons incubated for
1 h with different concentrations of tutin showed an increase in
phosphorylated CREB (Fig. 2D, n=3). For example, the
increase in CREB was concentration-dependent with values of
12±1% for 200 μM and 18±2% for 500 μM tutin (n=3).
In the present study, we analyzed the inhibitory effects of
tutin isolated from C. ruscifolia subspecie ruscifolia, a native
poisonous Chilean shrub, on inhibitory glycine receptors and its
impact on neuronal and network excitability in cultured spinal
neurons. Tutin was able to block glycine receptors in a
concentration-dependent manner and this effect was accompa-
nied by a marked increase in neuronal excitability, reflected by
changes in the spontaneous synaptic activity and associated to
an increase in the frequency of AMPAergic events. The increase
in neuronal activity was likely due to a blockade on both
glycine- and GABAA-mediated inhibitory currents by tutin.
Interestingly, we found two differences between tutin and
corymine, a toxin extracted from the leaves of Hunteria
zeylanica (Leewanich et al., 1997). First, the effect of tutin
was mainly non-competitive in glycine receptors. Second, tutin
was more effective in GABAA receptors than corymine
(Leewanich et al., 1997). In addition, we found an augmentation
in calcium transients in parallel with an increase in the activated
form of CREB, both being indicators of neuronal plasticity and
epileptogenic activity (Josselyn and Nguyen, 2005).
In conclusion, tutin appears to play an important role in the
toxic effects of the Coriariaceae family by inhibiting glycine
receptors in spinal neurons. In addition, it is likely that tutin also
induced toxic effects by inhibiting GABAAreceptors.
We thank Mrs. L. J. Aguayo and C. Lopez for their technical
assistance. This work was supported by the “Programa Bicente-
nario de Insercion de Postdoctorales en la Academia PBCT Nro.
7” (CONICYT-World Bank) and Proyecto DIUC-Patagonia
(University of Concepcion). Patagonian Ecosystems Research
1060368 and Innova BioBio 05B1397L7.
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