1Laboratori de Neurobiologia Cellular i Molecular, Departament de Biologia Cellular i Anatomia Patològica, Facultat de Medicina, Hospital de Bellvitge, Universitat de Barcelona, E-08907 L'Hospitalet de Llobregat, Spain; 2Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom; and 3Mayo Clinic Cancer Center and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905
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ABSTRACT |
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Ros, Esteve,
Jordi Aleu,
Inmaculada Gomez De
Aranda,
Carles Cantí,
Yuan-Ping Pang,
Jordi Marsal, and
Carles Solsona.
Effects of Bis(7)-Tacrine on Spontaneous Synaptic Activity and on
the Nicotinic ACh Receptor of Torpedo Electric Organ.
J. Neurophysiol. 86: 183-189, 2001.
Bis(7)-tacrine is a potent acetylcholinesterase inhibitor in which two
tacrine molecules are linked by a heptylene chain. We tested the
effects of bis(7)-tacrine on the spontaneous synaptic activity.
Miniature endplate potentials (MEPPs) were recorded extracellularly on
slices of electric organ of Torpedo marmorata. Bis(7)-tacrine, at a concentration of 100 nM, increased the magnitudes that describe MEPPs: amplitude, area, rise time, rate of rise, and
half-width. We also tested the effect of bis(7)-tacrine on nicotinic
acetylcholine receptors by analyzing the currents elicited by
acetylcholine (100 µM) in Torpedo electric organ membranes transplanted in Xenopus laevis oocytes. Bis(7)-tacrine
inhibited the acetylcholine-induced currents in a reversible manner
(IC50 = 162 nM). The inhibition of nicotinic
acetylcholine receptors was not voltage dependent, and bis(7)-tacrine
increased the desensitization of nicotinic acetylcholine receptors. The
Hill coefficient for bis(7)-tacrine was 0.72 ± 0.02, indicating
that bis(7)-tacrine binds to the nicotinic acetylcholine receptor in a
molecular ratio of 1:1, but does not affect the binding of
-bungarotoxin with the nicotinic acetylcholine receptor. In
conclusion, bis(7)-tacrine greatly increases the spontaneous quantal
release from peripheral cholinergic terminals at a much lower
concentration than tacrine. Bis(7)-tacrine also blocks
acetylcholine-induced currents of Torpedo electric organ,
although the mechanism is different from that of tacrine:
bis(7)-tacrine enhances desensitization, whereas tacrine reduces it.
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INTRODUCTION |
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Alzheimer's disease is the most
frequent cause of dementia in western populations over the age of 65. The drugs that have been most extensively studied for the palliative
treatment of Alzheimer's disease are acetylcholinesterase (AChE)
inhibitors (Krall et al. 1999), which, according to the
cholinergic hypothesis (Bartus et al. 1982
), increase
the levels of brain acetylcholine (ACh) and relieve cognitive
deficiencies. In the European Union, the only drugs approved for
clinical treatment are tacrine, donepezil, and rivastigmine, all of
which are AChE inhibitors (Francis et al. 1999
). Tacrine
was one of the first anticholinesterasic drugs approved by the Food and
Drug Administration of the United States in 1993 (Krall et al.
1999
). Doses between 80 and 160 mg per day are effective
(Farlow et al. 1992
; Knapp et al. 1994
),
but in some cases this concentration is hepatotoxic (Watkins et
al. 1994
). In addition, it has been demonstrated that tacrine
can induce myopathy by the excessive stimulation of nicotinic receptors
(Jeyarasasingam et al. 2000
).
Recently, it has been shown that tacrine could have other effects on
Alzheimer's disease that are not directly related to its AChE
inhibitory activity: 1) tacrine inhibits the secretion of
amyloid beta-peptides in human neuroblastoma cells (Lahiri et
al. 1997, 1998
); 2) in murine
neuroblastoma cells, acute application of tacrine inhibits monoamine
oxidase isoform A and activates the B isoform in the long term
(Zatta et al. 1998
); and 3) tacrine also
attenuates the influx of calcium by inhibiting the L-type calcium
channels (Dole
al et al. 1997
).
In the early 1990s, a number of second-generation AChE inhibitors were
developed, some of them based on the chemical structure of the tacrine
molecule. Bis(7)-tetrahydroaminacrine [bis(7)-tacrine; Fig.
1] is a potent and selective inhibitor
of AChE, in which two tacrine molecules are linked by a heptylene chain
spaced so as to permit simultaneous binding at the catalytic and
peripheral sites of AChE (Pang et al. 1996).
Bis(7)-tacrine is up to 150-fold more potent and 250-fold more
selective in inhibiting AChE than tacrine over butyrylcholinesterase
(BChE), and its effects on AChE are reversible (Wang et al.
1999
). Recent studies have also shown that bis(7)-tacrine
effectively reverses AF64A-induced deficits in navigational memory in
rats (Liu et al. 2000
) and is a potent GABA(A)-receptor
antagonist (Li et al. 1999
).
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Here we show the effect of bis(7)-tacrine molecule on the spontaneous
synaptic activity and on the current associated with the nicotinic
acetylcholine receptor. We used the electric organ of Torpedo
marmorata. Synaptic activity was recorded extracellularly on
slices of fresh electric organ (Cantí et al.
1994; Ros et al. 2000
). The activation of
nicotinic acetylcholine receptors was measured on Xenopus
laevis oocytes that were transplanted with membranes isolated from
the electric organ (Cantí et al. 1998
;
Marsal et al. 1995
; Ros et al. 2000
).
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METHODS |
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Animals and solutions
Torpedo marmorata specimens were caught off the Catalan Mediterranean coast and kept in artificial seawater. The fish were anesthetized with tricaine (3-aminobenzoic acid ethyl ester methanesulfonate salt; Sigma, St. Louis, MO) at a concentration of 0.03% in seawater, before surgical excision of electric organs.
Torpedo marmorata electric organ fragments were kept in the following saline solution (in mM): 280 NaCl, 3 KCl, 3.4 CaCl2, 1.8 MgCl2, 5.5 glucose, 300 urea, 100 sucrose, and 6.8 HEPES/NaOH-buffer, pH adjusted to 7.0 with NaHCO3. The same solution was used to record the spontaneous synaptic activity.
Mature females of Xenopus laevis were purchased from the Center d'Elevage des Xenopes (Montpellier, France) and were anesthetized by immersion in water containing 0.17% tricaine. A few lobes of ovaries were removed through a small incision in the abdomen.
Solutions for Xenopus oocytes were as follows. Barth's solution contained (in mM) 88 NaCl, 1 KCl, 0.33 Ca(NO3)2, 0.82 MgSO4, 2.40 NaHCO3, and 10 mM HEPES at pH 7.4, supplemented with 100 IU/ml penicillin and 0.1 mg/ml streptomycin. Recording solution contained (in mM) 115 NaCl, 2 KCl, 1.8 CaCl2, and 5 HEPES at pH 7.0. None of the Xenopus female donors used in this study exhibited muscarinic acetylcholine receptors in their oocytes.
Recording of the spontaneous synaptic activity
All recordings were made at room temperature (20-22°C). From 5 to 10 prisms of the electric organ were cut with a scalpel blade, and 1- to 5-mm sections were incubated overnight, in Torpedo saline solution containing bis(7)-tacrine, to ensure complete diffusion throughout the tissue. Measurements were performed in fragments fixed in a Plexiglas chamber with a silicone elastomer (Sylgard)-coated base.
The spontaneous synaptic release of ACh was recorded with focal
extracellular low-resistance microelectrodes (Katz and Miledi 1977) as adapted to electric organ by Soria
(1983
; see Dunant and Muller 1986
;
Muller and Dunant 1987
, for details) and as described elsewhere (Cantí et al. 1994
; Ros et al.
2000
). The method allows long-term recording with little damage
to the cells. Spontaneous miniature endplate potentials (MEPPs) were
amplified (Axoclamp-2A, Axon Instruments, Foster City, CA)
monitored on a Tektronix (Beaverton, OR) 5110 oscilloscope and recorded
in parallel on a VCR (Biologic, Echirolles, France) and on a
PC-Computer with a LabView (National Instruments, Austin, TX) program
(Quantadat) written in our laboratory with an AT-MIO16X (National
Instruments) digitizing interface. Signals were acquired at a frequency
of 100 kHz and analyzed using the same Labview program and the Whole
Cell Analysis program, kindly provided by Prof. J. Dempster
(Strathclyde University, Scotland, UK), and a TL-1 Labmaster digitizing
interface. Data in ASCII form were exported to Sigmaplot 4.01.
The following parameters of each MEPP were measured: amplitude, rise
time, rate or velocity of rising, the area corresponding to the charge
that generated them (measured as the integral of the contour delimited
by each MEPP), and the half-width that indicates the rate of the decay
phase (see Fig. 2A). Results
were obtained from five separate experiments, and they were represented
as histogram and cumulative plots (Van der Kloot 1991),
comparing all the variables in the different experimental conditions
(Fig. 3). The number of MEPPs analyzed
was 7,011 for the control condition and 7,290 for bis(7)-tacrine.
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Oocyte preparation, microinjection, and recording
Oocytes at stages V and VI (Dumont 1972) were
dissected out and kept at 15-16°C in sterile Barth's solution. One
day before injection the oocytes were treated with collagenase type 1A
(Sigma; 0.5 mg/ml) for 45-50 min at room temperature to remove the
surrounding layers (Miledi and Woodward 1989
).
Healthy oocytes were microinjected with 50 nl of thawed
suspension (2-8 mg/ml) of electroplaque membranes
(Cantí et al. 1998; Marsal et al.
1995
; Ros et al. 2000
) by means of an injector
(World Precision Instruments, Sarasota, FL, model A203XVZ). Samples
were sonicated prior to injection.
Eighteen to 48 h after the injection, oocytes were voltage clamped
with a two-electrode system (Axoclamp-2A, Axon Instruments). Intracellular electrodes (1-4 M resistance) were filled with 3 M
potassium chloride for voltage recording and with 3 M potassium acetate
for current injection. The volume of the oocyte recording chamber was
200 µl. Membrane currents were low-pass filtered at 10 Hz and
recorded on a PC using Whole Cell Analysis v. 2.1. program after
sampling signals by Lab PC+ (National Instruments) at twice the filter
frequency. In all recordings currents were elicited by challenges of
100 µM ACh chloride. The interval between consecutive responses was
systematically set to 10 min (flow rate 8 ml/min), as we had previously
established that this was sufficient to ensure a complete recovery from
receptor desensitization. All the oocytes were tested for consistent
response amplitudes, with at least three challenges prior to the
application of the drug.
125I--bungarotoxin binding
Binding of 125I--bungarotoxin (Amersham
Life Science, Piscataway, NJ) was undertaken according to the procedure
previously described (Schmidt and Raftery 1973
). In
brief, Torpedo electric organ membranes were solubilized for 30 min in
the following solution: 10 mM HEPES, 100 mM NaCl, and 1% Triton X-100
at pH 7.4 and centrifuged 30 min at 100,000 g. The protein
concentration of the supernatant was between 0.8 and 1.1 mg/ml.
Aliquots of 50 µl of the supernatant were incubated with
-bungarotoxin (Sigma) at 10 µg/ml and containing 2.5 µCi. In
some samples, bis(7)-tacrine at different concentrations was added.
After 2 h of incubation at room temperature, the mixture was
placed in Whatman DE-81 of 2.3-cm-diam disk and rinsed three times in
10 ml of 10 mM sodium phosphate buffer, 50 mM Na Cl, and 100 0.1%
Triton X at pH 7.4. Dried disks were counted in a 1470 Wizard
counter. Unspecific binding was subtracted.
Calculations and statistics
Differences between distribution functions were evaluated with Sigmastat 3.2 software (SPSS, Chicago, IL) by Mann-Whitney rank sum test. Dose-response data were fitted by a nonlinear regression to a sigmoidal curve using the Sigmaplot 4.01 (SPSS) and Inplot (GraphPad, San Diego, CA) programs. Values are expressed as means ± SE calculated by the program.
Drugs
The stock solution of bis(7)-tacrine (1 mM) was made with
deionized water and kept at 80°C before it was diluted to its final concentration in the recording solution.
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RESULTS |
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Effects on MEPPs
We have tested bis(7)-tacrine at a concentration of 10 nM on
fragments of electric organ from Torpedo marmorata, with
extracellular recording through a loose patch configuration
(Dunant and Muller 1986), and we did not detect any
change in the size of spontaneous MEPPs (data not shown). However, at
the concentration of 100 nM, bis(7)-tacrine increased all the
parameters of MEPPs (Fig. 2, B and C).
The amplitude of MEPPs in the untreated fragments (n = 7,011) was 1.27 ± 0.03 (SE) mV, whereas in the fragments treated with bis(7)-tacrine at the concentration of 100 nM (n = 7,290), it was 1.91 ± 0.02 mV. The distribution amplitude is shown in Fig. 3A.
The area under the profile of each MEPP reflects the electrical charge carried during the release of a single quantum of ACh. The area was 1.05 ± 0.01 mV · ms, in the untreated fragments, and it was 2.30 ± 0.03 mV · ms in the treated fragments. Bis(7)-tacrine increased the area of MEPPs in the untreated fragments by more than twofold. Figure 3B shows the statistical distribution of areas.
The rise time of an MEPP is the result of the addition of time periods from different cellular processes: the release and diffusion of ACh throughout the synaptic cleft and the opening time of the nicotinic acetylcholine receptors. The rise time of MEPP was 0.44 ± 0.01 ms in the untreated fragments and 0.48 ± 0.01 ms in the treated fragments. See Fig. 3C for statistical distribution.
The rate of rise between 10 and 90% of the MEPP amplitude was 3.29 ± 0.05 mV/ms in untreated fragments, while in the fragments treated with bis(7)-tacrine at the concentration of 100 nM it was 4.69 ± 0.08 mV/ms. Figure 3D shows the statistical distribution.
The duration or the width of an MEPP depends on the persistence of ACh within the synaptic cleft. The drugs with AChE inhibitory properties prolonged the action of ACh by decreasing its rate of hydrolysis, and the decay phase of the MEPPs was therefore prolonged, because a single molecule of ACh can interact more than once with nicotinic acetylcholine receptors. Since the decay phase of an MEPP is an exponential function, the end of which is difficult to establish, the half-width of an MEPP is particularly appropriate for estimating the duration of an MEPP and, thus very convenient for investigating the action of AChE inhibitory agents on synaptic activity. The half-width was 0.70 ± 0.01 ms in the untreated fragments and 0.87 ± 0.01 ms in the treated fragments. The half-width distribution is shown in Fig. 3E.
In all the variables analyzed, the differences between the two groups were significant (P < 0.001).
Effects on nicotinic acetylcholine receptors in transplanted Xenopus oocytes
Bis(7)-tacrine reduced the amplitude of currents elicited by 100 µM ACh in a dose-dependent manner (Fig.
4A). The inhibition was fully
reversible after a wash time of 10 min. A sigmoidal function of four
parameters fitted the curves representing the inhibitory action of
bis(7)-tacrine and tacrine. The IC50 for bis(7)-tacrine was 162 ± 5 nM, while for tacrine it was 8.9 ± 1.2 µM (tacrine data were taken from previous studies conducted in
our laboratory: Cantí et al. 1998). The Hill
coefficient for bis(7)-tacrine was
0.72 ± 0.02.
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The effect of bis(7)-tacrine on the amplitude of ACh-activated currents
in oocytes was not voltage dependent. In a series of experiments,
currents were elicited by 100 µM ACh at different holding potentials
(from 70 to +10 mV) in each oocyte. The concentration of
bis(7)-tacrine used (100 nM) was close to its
IC50 on nicotinic acetylcholine receptors. At
negative membrane potentials, the slope conductance of ACh currents in
the presence of bis(7)-tacrine was similar to control (peak current
reduction of 40 ± 5% at
70 mV compared with 46 ± 6% at
20 mV, n = 8; Fig.
5A). However, the reversal
potential of the ACh current was not modified by the treatment with
bis(7)-tacrine, showing that the ion selectivity of the channel was
maintained.
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Oocytes were exposed to solutions containing either ACh (100 µM) in
the presence or absence of bis(7)-tacrine (100 nM) during long periods
of time (30 s; see Fig. 5B). A prolonged exposure of the
nicotinic acetylcholine receptor to ACh results in a desensitization or
open channel block, which would perhaps be increased by bis(7)-tacrine. The time constant () was calculated by fitting the decay phase of
the ACh currents to a single exponential function. In oocytes stimulated with ACh in the absence of bis(7)-tacrine, the time constant
was
= 11.3 ± 4.1 s, while in the presence of
bis(7)-tacrine the time constant was reduced to
= 2.8 ± 0.3 s.
Effects of bis(7)-tacrine on the binding
of 125I--bungarotoxin to solubilized
electric organ membranes
The number of molecules of -bungarotoxin that bound to
solubilized membranes of Torpedo electric organ was
7.47.1012, which correspond to a concentration
about 1 nmol of
-bungarotoxin/mg of protein. We have found that
bis(7)-tacrine at concentrations of 10, 100, and 1,000 nM did with not
interfere the binding of
-bungarotoxin to the nicotinic
acetylcholine receptor (n = 3; Fig.
6).
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DISCUSSION |
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The clinical goal of the administration of AChE inhibitors is to
achieve high brain ACh levels and relieve the cognitive deficiency symptoms in Alzheimer's disease patients. Bis(7)-tacrine is a highly
potent second-generation AChE inhibitor. The lethal toxicity of
bis(7)-tacrine determined by intragastric administration to rats is
reportedly increased only twofold respect to tacrine (Han et al.
2000), yet the inhibition of AChE by bis(7)-tacrine and the
increase of acetylcholine spontaneous synaptic activity by bis(7)-tacrine described here are several order of magnitudes greater
than those reported for tacrine. Furthermore, bis(7)-tacrine is 24-fold
more potent in reversing ethylcholine mustard aziridinium ion-induced
memory impairment in rats (Liu et al. 2000
) and has a
much higher intragastric efficacy in inhibiting AChE (Wang et al. 1999
) than that shown by tacrine. It would therefore appear logical to expect bis(7)-tacrine to be less toxic than tacrine.
Physostigmine, tacrine (Cantí et al. 1994), and
the derivative of tacrine CI-1002 (Ros et al. 2000
)
increased the amplitude and duration of MEPPs recorded from the
electric organ of Torpedo. Increased amplitude of MEPPs were
also obtained on the neuromuscular junction using tacrine (Braga
et al. 1991
; Thesleff et al. 1990
) and CI-1002
(Ros et al. 2000
). The concentrations assayed for these
drugs were usually between 1 and 100 µM. In contrast, bis(7)-tacrine increased the size of MEPPs in the electric organ of Torpedo
at a much lower concentration (100 nM). This observation is consistent with reports that bis(7)-tacrine is a more potent inhibitor of AChE
than tacrine (Pang et al. 1996
; Wang et al.
1999
).
The prolongation of the decay phase of the MEPPs is directly related to
the AChE inhibitory activity. In previous studies (Cantí
et al. 1994; Ros et al. 2000
) we have found that
tacrine (100 µM) or CI-1002 (1 µM) prolonged the half-width and,
thus the time course of a single MEPP. However, the concentration of bis(7)-tacrine used in the present study was 1,000- to 10-fold lower
than those cited previously. These low concentrations of bis(7)-tacrine
produced a modest increase in the decay time in MEPPs with respect to
other AChE inhibitors. This effect may be related to a direct
interaction of bis(7)-tacrine on nicotinic acetylcholine receptor as
discussed below.
The increase in the rise time and the rate of rise of the MEPPs recorded on fragments of electric organ of Torpedo treated with bis(7)-tacrine suggests that, in addition to its AChE inhibitory activity, bis(7)-tacrine may affect the time course of exocytosis of a single synaptic vesicle.
Using membranes from the electric organ of Torpedo
transplanted in Xenopus oocytes, we tested the effects of
tacrine and bis(7)-tacrine on the native form of the nicotinic
acetylcholine receptor. Membrane transplantation preserves the activity
of native receptors much better than the receptors translated in
oocytes from mRNA (Marsal et al. 1995; Morales et
al. 1995
). The IC50 for ACh-induced
currents of bis(7)-tacrine (162 nM) was much lower than previously
reported for tacrine (8.9 µM) (Cantí et al.
1998
), which indicates that bis(7)-tacrine is a more potent
inhibitor of nicotinic acetylcholine receptor than tacrine.
Consequently, bis(7)-tacrine needs to recruit more ACh molecules for
release by the exocytosis of a single vesicle to increase the size of
an MEPP, thus overcoming the higher inhibition of the nicotinic
acetylcholine receptor.
The Hill coefficient suggested that bis(7)-tacrine, like tacrine, binds
to the nicotinic acetylcholine receptor in a molecular ratio of 1:1.
However, in contrast to tacrine (Cantí et al.
1998), the action of bis(7)-tacrine was not sensitive to
membrane voltage, suggesting that the drug did not interact preferably
with the open-state receptor. According to the records obtained during long periods of ACh perfusion, the receptor desensitized more quickly
in the presence of bis(7)-tacrine, suggesting that the drug-receptor
complex closed faster than in the absence of the drug. It might be the
case that part of this effect is caused for the open channel block by
the drug. Our results indicate that the union of the bis(7)-tacrine to
the nicotinic acetylcholine receptor must accupy a different position
within the
-bungarotoxin site, perhaps interacting with the inner
part of the receptor. These results contrast with those obtained with
tacrine on nicotinic acetylcholine receptor desensitization
(Cantí et al. 1998
), in which tacrine slowed
down receptor desensitization. These results also suggest that
bis(7)-tacrine might act as a slowly dissociating antagonist of the
nicotinic acetylcholine receptor, whereas tacrine would be a rapidly
dissociating antagonist (due to its lower affinity). If the latter were
the case, part of the tacrine molecules unbound from the nicotinic
acetylcholine receptors would be replaced by ACh molecules, giving the
effect of a slowed desensitization (Clements 1996
).
In summary, bis(7)-tacrine produced similar effects to those of tacrine
on spontaneous synaptic activity, but at concentrations that were
100-1,000 times lower. The action of bis(7)-tacrine on nicotinic
acetylcholine receptors differs from that of tacrine. Even if we
consider that the cholinergic synapse of the electric organ is not the
same as the central nervous cholinergic synapse, our results suggest
that bis(7)-tacrine should have beneficial effects on cholinergic
function in the CNS at a much lower concentration than tacrine, and the
potency of bis(7)-tacrine in proportion with its toxicity may therefore
be less toxic in the palliative treatment of Alzheimer's disease
patients (Han et al. 2000). On the basis of the present
study, the reported biochemical, pharmacological, and behavioral
information (Li et al. 1999
; Liu et al.
2000
; Pang et al. 1996
; Wang et al.
1999
), and the low-cost synthesis of bis(7)-tacrine
(Pang et al. 1996
), it appears that bis(7)-tacrine fits
closely with the established criteria for an ideal cholinergic drug for
the palliative treatment of Alzheimer's disease.
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ACKNOWLEDGMENTS |
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We thank Dr. J. Dempster for kindly providing "Whole Cell Analysis Program 2.1." We are also indebted to J. Segura, Universitat Politècnica de Catalunya, for writing the Labview software (Quantadat) to record and analyze MEPPs, and Servei d'Assesorament Lingüístic of the University of Barcelona.
This study was supported by Dirección General de Enseñanza Superior e Investigación Científica from the Spanish Government and Comissió Interdepartamental de Recerca i Innovació Tecnològica from the Generalitat de Catalunya.
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FOOTNOTES |
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Address for reprint requests: C. Solsona, Laboratori de Neurobiologia Cellular i Molecular, Departament de Biologia Cellular i Anatomia Patològica, Facultat de Medicina, Hospital de Bellvitge, Universitat de Barcelona, Campus de Bellvitge, Pavelló de Govern, Feixa Llarga s/n, E-08907 L'Hospitalet de Llobregat, Spain (E-mail: solsona{at}bellvitge.bvg.ub.es).
Received 5 June 2000; accepted in final form 23 March 2001.
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REFERENCES |
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