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Vol. 61, Issue 5, 1222-1234, May 2002
Department of Pharmacology and Experimental Therapeutics (M.D.S., M.A., E.F.R.P., A.M., E.X.A.) and Department of Neurosurgery (H.M.E.), University of Maryland School of Medicine, Baltimore, Maryland; Departmento de Farmacologia Básica e Clínica, Instituto de Ciênicas Biomédicas (Y.A., E.X.A.) and Instituto de Biofísica Carlos Chagas Filho (M.D.S.), Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; and Institute of Physiological Chemistry and Pathobiochemistry, Johannes-Gutenberg University Medical School, Duesbergweg, Mainz, Germany (A.M.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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In this study, the patch-clamp technique was used to determine the
effects of galantamine, a cholinesterase inhibitor and a nicotinic
allosteric potentiating ligand (APL) used for treatment of Alzheimer's
disease, on synaptic transmission in brain slices. In rat hippocampal
and human cerebral cortical slices, 1 µM galantamine, acting as
a nicotinic APL, increased 
-aminobutyric acid (GABA) release
triggered by 10 µM acetylcholine (ACh). Likewise, 1 µM galantamine,
acting as an APL on presynaptically located nicotinic receptors
(nAChRs) that are tonically active, potentiated glutamatergic or
GABA-ergic transmission between Schaffer collaterals and CA1 neurons in
rat hippocampal slices. The cholinesterase inhibitors rivastigmine,
donepezil, and metrifonate, which are devoid of nicotinic APL action,
did not affect synaptic transmission. Exogenous application of ACh
indicated that high and low levels of nAChR activation in the Schaffer
collaterals inhibit and facilitate, respectively, glutamate release
onto CA1 neurons. The finding then that the nAChR antagonists
methyllycaconitine and dihydro-
-erythroidine facilitated
glutamatergic transmission between Schaffer collaterals and CA1 neurons
indicated that in a single hippocampal slice, the inhibitory action of
strongly, tonically activated nAChRs in some glutamatergic fibers
prevails over the facilitatory action of weakly, tonically activated
nAChRs in other glutamatergic fibers synapsing onto a given neuron.
Galantamine is known to sensitize nAChRs to activation by low, but not
high agonist concentrations. Therefore, at 1 µM, galantamine is
likely to increase facilitation of synaptic transmission by weakly,
tonically activated nAChRs just enough to override inhibition by
strongly, tonically activated nAChRs. In conclusion, the nicotinic APL
action can be an important determinant of the therapeutic effectiveness
of galantamine.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Alzheimer's
disease (AD) is a neurodegenerative disorder that afflicts millions
worldwide. It is characterized by a progressive decline of intellectual
abilities, which eventually becomes severe enough to interfere with
social or occupational individual functioning and ultimately leads to
death (Chung and Cummings, 2000
). Although the cause of the disorder
remains unknown, there is a strong correlation between brain
cholinergic dysfunction and the severity of AD symptoms (Coyle et al.,
1983; Nordberg, 1999). Post-mortem biopsy studies have shown that as
the condition worsens, progressive loss of basal forebrain cholinergic
neurons, which innervate the entire cortical mantle (Cullen et al.,
1997), is accompanied by incremental loss of nAChRs in cerebral
cortical neurons (Nordberg, 1999; Perry et al., 2000). Therefore,
increasing brain nicotinic functions to a level sufficient to improve
synaptic plasticity and neuronal survival emerges as a promising
therapeutic approach for treatment of AD patients.
Two nAChR subtypes are found in abundance in the mammalian central
nervous system (CNS). One binds nicotine with high affinity and is
composed of 
4- and 2-subunits; the other binds -bungarotoxin and is most probably a homomeric 7-nAChR (Lindstrom, 1997). These receptors are located postsynaptically, where they mediate fast synaptic transmission (Albuquerque et al., 2000a), and presynaptically, where they modulate synaptic transmission mediated by numerous neurotransmitters, including glutamate, GABA, serotonin, ACh, and
noradrenaline (Albuquerque et al., 2000b).
At the neuromuscular junction, cholinergic nicotinic function can be
enhanced by cholinesterase inhibitors and nicotinic agonists (Taylor,
1982). In the nervous systems, however, the effects of these agents are
more complex. First, neuronal nAChRs, particularly 7-nAChRs, are
much more prone to agonist-induced desensitization than muscle nAChRs.
Thus, nicotinic agonists only transiently increase nicotinic function
in CNS neurons (Alkondon et al., 2000b). Furthermore, unlike
muscle nAChRs, some neuronal nAChRs, including 7-nAChRs, recognize
both ACh and its metabolite choline as full agonists; the
EC50 values for ACh and choline as 7-nAChR
agonists are approximately 140 µM and 1.6 mM, respectively
(Albuquerque et al., 2000b). Therefore, cholinesterase inhibition may
not necessarily enhance functions mediated by these nAChRs. In fact,
cholinesterase inhibitors do not affect 7-nAChR-mediated synaptic
transmission evoked by low-frequency stimulation of cholinergic fibers
in chick ciliary ganglia (Zhang et al., 1996).
An alternative means to increase nicotinic functions in the brain is to
"sensitize" the nAChRs to activation by the endogenous agonist(s).
In the middle 1980s, the anticholinesterase physostigmine was shown to
activate frog muscle nAChRs (Shaw et al., 1985). Subsequent studies not
only confirmed the nicotinic agonistic activity of physostigmine, but
also demonstrated that such an effect is insensitive to blockade by
classical nicotinic antagonists (Okonjo et al., 1991; Pereira et al.,
1993). The agonistic activity of physostigmine-like compounds,
initially referred to as "noncompetitive agonists" (Storch et al.,
1995), is the result of their binding to a site close to, but distinct
from, the ACh-binding site on nAChR -subunits (Schrattenholz et al.,
1993). Noncompetitive agonists are weak agonists; by themselves, they
cannot induce macroscopic nicotinic responses (Pereira et al., 1994;
Storch et al., 1995). They can, however, potentiate the nAChR activity induced by classical nAChR agonists, and are, therefore, also referred
to as nicotinic APLs (Maelicke and Albuquerque, 1996; Schrattenholz et
al., 1996).
Galantamine, an alkaloid originally obtained from bulbs of snowdrops,
is a weak cholinesterase inhibitor and a powerful nicotinic APL that
seems to be more effective and less toxic than most cholinesterase inhibitors currently used to treat AD patients (Woodruff-Pak et al.,
2001). However, the effects of galantamine on neuronal functions in the
CNS and the relevance of its nicotinic APL action remain elusive. Thus,
the present study was designed to investigate whether galantamine
affects synaptic transmission and, if so, by what mechanism. Evidence
is provided that galantamine, acting as an APL on presynaptically
located nAChRs that are weakly, tonically activated, induces
long-lasting facilitation of glutamatergic or GABA-ergic transmission
between the Schaffer collaterals and CA1 neurons in rat hippocampal
slices. Such an effect is also observed when hippocampal slices are
exposed to the anticholinesterase and nicotinic APL methyl-galantamine,
but is not observed when the slices are exposed to cholinesterase
inhibitors devoid of nicotinic APL action, including methamidophos,
donepezil, rivastigmine, and metrifonate (Fig.
1). Furthermore, via its nicotinic APL
action, galantamine potentiates GABA release triggered by low ACh
concentrations exogenously applied to rat hippocampal and human
cerebral cortical slices. Taken together these results indicate that
the nicotinic APL action can contribute to the cognitive improvement
observed in AD patients treated with galantamine.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Rat Hippocampal Slices.
Slices of 250-µm thickness were
obtained from the hippocampi of 15- to 25-day-old Sprague-Dawley rats
according to the procedure described previously (Alkondon et al.,
1999). The slices were stored in a holding chamber containing
artificial cerebrospinal fluid (ACSF) bubbled with 95%
O2 and 5% CO2 and were
maintained at room temperature. Each slice, as needed, was transferred
to a recording chamber (capacity of 2.0 ml) and held submerged by two
nylon fibers. The recording chamber was continuously perfused with
bubbled ACSF, which had the following composition: 125 mM NaCl, 25 mM
NaHCO3, 2.5 mM KCl, 1.25 mM
NaH2PO4, 2 mM
CaCl2, 1 mM MgCl2, and 25 mM glucose (osmolarity ~ 340 mOsM).
Cultured Hippocampal Neurons.
Primary cultures were prepared
from the hippocampi of 16- to 18-day-old fetal Sprague-Dawley rats
according to the procedure described elsewhere (Pereira et al., 1993).
Human Cerebral Cortical Slices.
Slices of 250-µm thickness
were prepared from specimens of the human lateral neocortex obtained
from the temporal or frontal cortical lobe of three male and three
female patients according to the procedure described by Alkondon et al.
(2000b).
Electrophysiological Recordings.
By means of the whole-cell
mode of the patch-clamp technique, postsynaptic currents (PSCs) were
recorded from neurons of the CA1 pyramidal layer of rat hippocampal
slices in response to afferent stimulation. Test solutions were applied
to the slices through a set of coplanar-parallel glass tubes (400 µm
i.d.) glued together and assembled on a motor driven system (Newport
Corporation, Irvine, CA) controlled by microcomputer. The tubes were
placed at a distance of approximately 100 to 150 µm from the slice,
and the gravity-driven flow rate was adjusted to 1.0 ml/min. Each tube
was connected to a different reservoir filled with test solution.
Evoked PSCs were recorded after application of a supramaximal 20- to
60-µs electrical stimulus via a bipolar electrode made of thin
platinum wires (50- to 100-µm diameter). The stimulus was delivered
by an isolated stimulator unit (Digitimer Ltd., Garden City, England) connected to a digital-to-analog interface (TL-1 DMA; Axon Instruments, Union City, CA). The platinum electrode was positioned in the stratum
radiatum approximately 250 to 300 µm away from the cell body of the
neurons in the CA1 pyramidal layer of the hippocampal slices. Possible
changes in series resistance were detected by applying online a
hyperpolarizing pulse (5 mV) before the stimulus pulse. Excitatory and
inhibitory postsynaptic currents (EPSCs and IPSCs, respectively) evoked
by field stimulation were pharmacologically identified and isolated by
the application of antagonists of the excitatory
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and
N-methyl-D-aspartate (NMDA) receptors,
20 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 µM
2-amino-5-phosphonovaleric acid (APV), respectively, or the antagonist
of the inhibitory GABAA receptor, 100 µM picrotoxin.
) electrodes were pulled from
borosilicate capillary glass (World Precision Instruments, New Haven,
CT) and filled with internal solution. The composition of the internal
solution used for voltage-clamp recordings from neurons in the CA1
pyramidal layer was: 80 mM CsCl, 80 mM CsF, 10 mM EGTA, 22.5 mM CsOH,
10 mM HEPES, and 5 mM QX-314 (pH adjusted to 7.3 with CsOH; 340 mOsM).
The composition of the internal solution used for recording from CA1
interneurons and from human cerebral cortical neurons was: 130 mM
Cs-methane sulfonate, 10 mM CsCl, 2 mM MgCl2,
5 mM QX-314, 10 mM EGTA, and 10 mM HEPES (pH adjusted to 7.3 with
CsOH; 340 mOsM). The internal solution for current-clamp recordings
from neurons in the CA1 pyramidal layer of rat hippocampal slices had
the following composition: 130 mM K-gluconate, 20 mM KCl, 10 mM
EGTA, and 10 mM HEPES (pH adjusted to 7.3 with KOH; 340 mOsM). In most
experiments, biocytin (0.5%) was included in the internal solution for
later identification of the neuron type. All experiments were performed
in the presence of the muscarinic receptor antagonist atropine (1 µM)
and at room temperature (20-22°C).
Data Analysis.
Peak amplitude, 10 to 90% rise time and
decay-time constant of field stimulation-evoked PSCs and of
spontaneously occurring action potentials were determined using the
pClamp6 software. Spontaneously occurring and ACh-triggered IPSCs were
analyzed using the Continuous Data Recording software (Dempster, 1989). Results are presented as means ± S.E.M. and were compared for their statistical significance using the Student's t test
or one-way ANOVA followed by Dunnett's test.
Drugs.
Galantamine HBr and methyl-galantamine Br were
provided by Boehringer Ingelheim GmbH (Ingelheim, Germany). A 100 mM
stock solution of methamidophos (99.8%; Bayer AG, Leverkussen,
Germany) was prepared and kept at 
20°C, and working dilutions were
made daily just before the experiments. Donepezil HCl and rivastigmine hydrogen tartrate were kindly provided by Prof. Madeleine M. Joullie and Michael S. Leonard (Department of Chemistry, University of Pennsylvania, Philadelphia). Janssen Research Foundation also provided
a pure sample of Donepezil HCl. Methyllycaconitine (MLA) citrate was a
gift from Prof. M. H. Benn (Department of Chemistry, University of
Calgary, Alberta, Canada). Dihydro--erythroidine (DHE)
hydrobromide was a gift from Merck (Rahway, NJ). All other chemicals were purchased from Sigma (St. Louis, MO). A 250 mM stock
solution of picrotoxin was made in dimethyl sulfoxide, and dilutions
were made in the ACSF. NaOH was used to dissolve CNQX and APV (the 10 mM stock solution of CNQX had 12.5 mM NaOH and the 50 mM stock solution
of APV had 0.5 M of NaOH). Donepezil and rivastigmine were dissolved in
DMSO and diluted further with ACSF.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effects of Galantamine on Glutamatergic Transmission in Rat
Hippocampal Slices: Time and Concentration Dependence.
In the
continuous presence of the GABAA receptor
antagonist picrotoxin (100 µM), inward PSCs were recorded at 60 mV
from neurons in the CA1 pyramidal layer of rat hippocampal slices in response to field stimulation of the Schaffer collaterals. These currents were glutamatergic in nature, because they were reversibly inhibited by exposure of the slices to 20 µM CNQX and 50 µM APV (Fig. 2A), and they are, herein, referred
to as EPSCs. After recording stable responses for at least 5 min under
control conditions, EPSCs were recorded during a subsequent 5-min
perfusion of the slices with ACSF containing 1 µM galantamine. At the
end of the 5-min exposure of the slices to galantamine, there was an
increase in the amplitude of EPSCs (Fig. 2A). The potentiating effect
of galantamine was reversible upon wash.
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Galantamine Does Not Alter the Activity of Postsynaptic Glutamate
Receptors.
The above results suggest that galantamine facilitates
glutamate transmission via a presynaptic mechanism of action. They do
not rule out, however, the possibility that galantamine also alters the
sensitivity of the postsynaptic glutamatergic receptors to glutamate.
To address this possibility, whole-cell currents evoked by 2-s pulses
of AMPA, kainate, or NMDA (each at 30 µM, a subsaturating
concentration) were recorded from cultured hippocampal neurons before
and during their perfusion with external solution containing 1 µM
galantamine. Galantamine was present in both the background perfusion
and the agonist solution. After a 10-min exposure of the neurons to 1 µM galantamine, there were no apparent changes in the amplitude or
kinetics of currents evoked by each agonist (Fig.
4, A and B).
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Galantamine on Membrane and Action Potentials in Hippocampal
Neurons.
Alterations in membrane and action potentials could also
account for the effects of galantamine on synaptic transmission; however, 5- to 10-min exposures of rat hippocampal slices to 1 or 10 µM galantamine had no significant effect on the membrane potential or
the peak amplitude, rate of rise, duration, and frequency of
spontaneously occurring action potentials recorded from CA1 pyramidal
neurons (Table 2). Furthermore,
galantamine caused no change in the input resistance of the neurons
(Table 2).
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Effects of Cholinesterase Inhibition and nAChR Activation on Field
Stimulation-Evoked EPSCs in Rat Hippocampal Slices.
To determine
the effect of cholinesterase inhibition on glutamatergic transmission,
field stimulation-evoked EPSCs were recorded from CA1 neurons before,
during, and after their exposure to methamidophos, a cholinesterase
inhibitor devoid of the nicotinic APL action (Camara et al., 1997; Fig.
1). Methamidophos was tested at concentrations that inhibit
cholinesterase activity in the hippocampus by approximately 10, 30, and
100% (i.e., 1, 10, and 100 µM) (Camara et al., 1997). No significant
changes in EPSC amplitudes were observed when the slices were exposed
to 1 and 10 µM methamidophos. An increase in the EPSC amplitude was
observed 30 s after exposure of the slices to 100 µM
methamidophos; however, this effect decreased with time, becoming
negligible after a 2-min exposure of the preparations to 100 µM
methamidophos (Fig. 5A). Because of the
transient nature of the effect of cholinesterase inhibition on
glutamatergic transmission, the net result of a 5-min exposure of the
slices to 1 to 100 µM methamidophos was no apparent change in the
amplitude of EPSCs (Fig. 5B; Table 1).
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Involvement of 7-nAChRs in Galantamine-Induced Potentiation of
Glutamatergic Transmission in Rat Hippocampal Slices.
To determine
the contribution of the different nAChR subtypes to regulation of
glutamatergic transmission between the Schaffer collaterals and neurons
in the CA1 pyramidal layer of the hippocampus, field stimulation-evoked
EPSCs were recorded from CA1 neurons before, during, and after
perfusion of hippocampal slices with ACSF containing the 7-nAChR
antagonist MLA (10 nM) and/or DHE, a nicotinic antagonist that at 10 µM reduces by ~40 and 90% the activity of 7- and
42-nAChRs, respectively (Alkondon et al., 1999). A 10-min
exposure of the slices to MLA increased by ~20% the amplitude of the
evoked EPSCs (Fig. 7A). Approximately
10% enhancement of the amplitude of evoked EPSCs was observed when the
slices were exposed for 10 min to DHE (Fig. 7A). Potentiation of
glutamatergic transmission by DHE was fully reversed after 10 min of
washing of the slices with DHE-free ACSF; the average amplitudes of
EPSC recorded after washing the preparations were 104 ± 2% of
those recorded under control conditions (n = 5 neurons). The effect of MLA was only partially reversed after 10 min of washing the preparations with MLA-free ACSF; the average EPSC amplitudes recorded after 10 min of washing the preparations were 108 ± 1% of those recorded under control conditions
(n = 5 neurons). The effects of DHE and MLA on
evoked EPSCs were not additive. In fact, the total effect of both
antagonists together was ~5% smaller than that of MLA alone and
~5% higher than that of DHE alone (Fig. 7A), suggesting a
competitive interaction between the two antagonists with the same
receptor (i.e., the 7-nAChR). Considering that neither MLA nor
DHE affects the postsynaptic glutamatergic receptors (Alkondon et
al., 1999), the present findings suggest that in a single hippocampal
slice the net effect of 7-nAChRs tonically activated to different
levels on Schaffer collaterals is a reduction of glutamate release onto
CA1 neurons.
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E
(see Fig. 7B), 1 µM galantamine did not alter glutamatergic transmission between the Schaffer collaterals and CA1 neurons in the
hippocampal slices. After 10 min of perfusion of the slices with ACSF
containing MLA or the admixture of MLA and DHE, the average EPSC
amplitudes were 121 ± 1% and 117 ± 5% of those recorded under control conditions (Fig. 7A). After an additional 5-min perfusion
of the slices with ACSF containing 1 µM galantamine in addition to
MLA or MLA-plus-DHE, the average EPSC amplitudes were 122 ± 1% and 115 ± 7%, respectively, of those recorded under control
conditions (n = 5 neurons). However, when the
7-nAChRs were only partially blocked by 10 µM DHE, the
potentiating effect of galantamine on glutamatergic transmission added
up to that of DHE. After a 10-min perfusion of hippocampal slices
with ACSF containing DHE, the average EPSC amplitudes were 110 ± 1% of those recorded under control conditions (n = 5 neurons). After a subsequent 5-min perfusion of the slices containing
galantamine in addition to DHE, the average EPSC amplitudes were
115 ± 1% of those recorded under control conditions
(n = 5 neurons); the additional 5% increase in the
EPSC amplitudes observed in the presence of galantamine was
statistically significant (p < 0.05 according to the
paired Student's t test).
Potentiation and inhibition of evoked EPSCs by 30 nM and 30 µM ACh,
respectively, could not be observed in slices that had been pre-exposed
for 10 min to the admixture of MLA and DHE (Fig. 7B). The fact that
the potentiating effect of galantamine and 30 nM ACh on evoked EPSCs
did not add up to that of the nicotinic antagonists could not be
explained by saturation of the system, because a 5-min exposure of the
preparations to the K+-channel blocker
4-aminopyridine (100 µM) increased the amplitudes of evoked EPSCs to
229 ± 14% of control (n = 3 neurons). These results support the concept that changes induced in the EPSCs by
exogenously applied galantamine and ACh were the result of the
interaction of these compounds with nAChRs.
One could argue that the potentiating effects of 30 nM ACh on evoked
EPSCs were the result of nAChR desensitization rather than activation.
However, MLA and DHE suppressed 30 nM ACh-induced enhancement of
EPSC amplitude when they were applied to the slices after the onset of
the potentiating effect of ACh. For example, the mean peak EPSC
amplitude recorded from six CA1 pyramidal neurons after a 5-min
exposure of the hippocampal slices to 30 nM ACh was 124.5 ± 5.1%
of that recorded under control conditions. During subsequent exposure
of the preparations to ACSF containing ACh and the antagonists, the
mean peak EPSC amplitude decreased significantly to 107.5 ± 4.1%
of control (p < 0.05 according to the one-way ANOVA)
returning to levels that were not statistically different from those
observed in the presence of the antagonists alone (i.e., 117 ± 5% of control; see Fig. 7A). Taken together these results indicate
that the effect of 30 nM ACh on evoked glutamatergic transmission is
the result of ACh-induced nAChR activation.
Effects of the Monoclonal Antibody FK-1 on Galantamine-Induced
Potentiation of Glutamatergic Transmission in Rat Hippocampal
Slices.
To investigate the mechanism underlying the effects of
galantamine on glutamatergic synaptic transmission, the effects of 1 µM galantamine on field stimulation-evoked EPSCs were analyzed before
and 30 min after perfusion of the hippocampal slices with ACSF
containing the monoclonal antibody FK-1 (1 µM). This antibody specifically recognizes the binding region of APLs on the -subunit of the nAChRs (Schrattenholz et al., 1993) and is a functional inhibitor of the noncompetitive agonistic (Pereira et al., 1993) and
allosteric potentiating actions (Schrattenholz et al., 1996) of
nicotinic APLs.
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Effects of Galantamine on GABA-ergic Transmission in the Rat
Hippocampus.
When 20 µM CNQX and 50 µM APV were both present
in the ACSF, inward PSCs were recorded at 60 mV from neurons in the
CA1 pyramidal layer of the hippocampal slices in response to field
stimulation of the Schaffer collaterals (Fig.
9A). These currents, which are herein
referred to as IPSCs, were GABA-ergic in nature because they were
inhibited by 100 µM picrotoxin (Fig. 9A).
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60 mV were 1.2 ± 0.25 Hz and 24.3 ± 2.9 pA, respectively. After a 10-min exposure to 1 µM galantamine, the mean frequency and amplitude of mIPSCs were 1.3 ± 0.34 Hz and
21.7 ± 4.2 pA, respectively. Also, the amplitudes of whole-cell currents evoked by 2-s pulses of 30 µM GABA in cultured hippocampal neurons exposed for 5 to 10 min to 1 µM galantamine were 97.5 ±3.8% of those of GABA-evoked whole-cell currents recorded before exposure of the neurons to the drug (n = 3 neurons).
These findings indicate that galantamine does not alter the activity of
postsynaptic GABAA receptors and suggest that
galantamine-induced potentiation of evoked IPSCs is the result of a
presynaptic action.
Effects of Galantamine on ACh-Triggered GABA-ergic IPSCs in Rat
Hippocampal Slices.
A different protocol was used to verify
whether the effects of galantamine on GABA-ergic transmission are
mediated via its interaction with nAChRs present on GABA-ergic neurons
synapsing onto the neurons from which recordings were obtained. In this protocol, a methanesulfonate-based internal solution was used and IPSCs
were selectively recorded from CA1 stratum radiatum interneurons
voltage-clamped at 0 mV. In the presence of muscarinic receptor
antagonist atropine, U-tube application of 10 µM ACh to the slices
enhanced the frequency of spontaneous IPSCs recorded from the
interneurons (Fig. 10A). Previous
studies have demonstrated that this effect of ACh is mediated by
activation of 7- and 42-nAChRs on GABA-ergic neurons synapsing
onto neurons from which recordings are obtained (Alkondon et al.,
1999).
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Effects of Galantamine on ACh-Triggered IPSCs in Human Cerebral
Cortical Slices.
To determine whether the effects of galantamine
on synaptic transmission observed in the rat preparation can be
extended to humans, experiments were performed in neurons of human
cerebral cortical slices according to techniques described previously
(Alkondon et al., 2000b). In the continuous presence of the muscarinic
receptor antagonist atropine (1 µM), U-tube application of 10 µM
ACh to neurons in human cerebral cortical slices triggered IPSCs (Fig. 11). This effect of ACh is the result
of its interaction with 42-nAChRs present on GABA-ergic neurons
synapsing onto the neurons from which recordings are obtained (Alkondon
et al., 2000b).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The present study demonstrates that galantamine facilitates both
excitatory and inhibitory transmissions in the rat and human brains and
that these effects are the result of the nicotinic APL action of
galantamine. The concentrations at which galantamine facilitates
synaptic transmission are very similar to those achieved in the brain
of rats treated with memory-enhancing doses of the drug (Bores et al.,
1996). Thus, facilitation of synaptic transmission by galantamine is
likely to underlie its therapeutic effectiveness in AD.
Galantamine Potentiates Glutamatergic Transmission by Increasing Glutamate Release via a Cholinesterase-Unrelated Mechanism. Galantamine (0.5-3 µM) enhanced the amplitude of EPSCs recorded from CA1 neurons in response to field stimulation of the Schaffer collaterals in rat hippocampal slices. The rapid onset and reversibility of the effect suggested that galantamine interacts with an extracellular target. Several lines of evidence indicated a presynaptic site of action for galantamine: 1) the drug had no effect on postsynaptic glutamatergic receptors; 2) the amplitudes of both AMPA/kainate and NMDA components of evoked EPSCs were increased by galantamine; and 3) the sum of the magnitude of the effects of galantamine on both components was equal to the magnitude of the drug's effect on compounded EPSCs. Although galantamine-induced potentiation of EPSCs was the result of a presynaptic action, it could not be explained by changes in Na+ and K+ conductances, because the drug caused no changes in the membrane properties of CA1 pyramidal neurons.
Even though the concentrations at which galantamine facilitates glutamatergic transmission are well within the range (0.4-4.0 µM) reported to inhibit by 50% brain cholinesterase activity (Sweeney et al., 1989; Bickel et al., 1991; Bores et al., 1996), the
anticholinesterase activity per se cannot explain the effect of
galantamine on glutamatergic transmission. First, long-lasting
facilitation of glutamate release by prolonged exposure (
5 min) of
hippocampal slices to galantamine could not be observed when the slices
were exposed to cholinesterase inhibitors devoid of nicotinic APL
activity, including methamidophos, metrifonate, donepezil, and
rivastigmine, each tested at nearly saturating concentrations for
cholinesterase inhibition. Second, the concentration dependence and
dynamics of the effects of methamidophos on evoked EPSCs differed
drastically from those of galantamine. Whereas the effects of
methamidophos on evoked EPSCs were only observed at concentrations that
inhibit by 100% cholinesterase activity (Camara et al., 1997), those
of galantamine were observed at concentrations that inhibit
cholinesterase activity by ~50%. In addition, galantamine-induced
facilitation of glutamatergic transmission lasted for as long as the
neurons were exposed to galantamine, whereas methamidophos-induced
potentiation of evoked EPSCs was transient, lasting no longer than 1 min. Third, the magnitude of galantamine's effect on evoked
EPSCs in hippocampal slices in which cholinesterase was blocked by
methamidophos was the same as that observed in control slices.
Galantamine-Induced Facilitation of Synaptic Transmission in
the Rat Hippocampus and the Human Cerebral Cortex Is Mediated by Its
Action as a Nicotinic APL.
In addition to increasing the amplitude
of evoked EPSCs, galantamine, via a presynaptic action, also increased
the amplitude of evoked IPSCs in the rat hippocampus and the frequency
of IPSCs triggered by low concentrations of ACh in the rat hippocampus and human cerebral cortex. As reported in previous studies, GABA-ergic transmission in the rat hippocampus and human cerebral cortex is
modulated by presynaptically located 7- and/or 42-nAChRs (Alkondon et al., 1999; 2000b). In this study, exogenous application of
nAChR antagonists (MLA and DHE) and different concentrations of ACh
to hippocampal slices led to the conclusions that 7-nAChRs are
present and tonically active in the Schaffer collaterals and that,
depending on the degree of receptor activity, glutamate release onto
CA1 neurons can be facilitated or inhibited (see Fig. 6).
7-nAChR activation was probably the
result of increased intracellular Ca2+
concentrations (Radcliffe et al., 1999; Albuquerque et al., 2000b). In
contrast, inhibition of synaptic transmission was probably the result
of 7-nAChR activation to a degree that causes enough depolarization
to inactivate Na+ channels and to dampen the
active propagation of action potentials (Alkondon et al., 2000a). The
finding that MLA and DHE potentiate glutamatergic transmission
between Schaffer collaterals and CA1 neurons indicated that, overall,
this transmission is preset by the inhibitory action of strongly,
tonically activated nAChRs in some glutamatergic fibers prevailing over
the potential facilitatory action of weakly, tonically activated nAChRs
in other glutamatergic fibers synapsing onto a CA1 neuron. Tonically
activated nAChRs are also known to modulate synaptic transmission in
other areas of the CNS (Cordero-Erausquin and Changeux, 2001).
The potentiating effect of galantamine on evoked glutamatergic
transmission in hippocampal slices is mediated by its interaction as an
APL with presynaptically located 7-nAChRs, because the effect is
observed in the presence of 7-nAChR antagonists or FK1, which
functionally antagonizes nicotinic APL actions (Pereira et al., 1993;
Schrattenholz et al., 1993, 1996). Considering that galantamine
sensitizes nAChRs to activation by low, but not high, agonist
concentrations (Schrattenholz et al., 1996; Samochocki et al., 2000),
it can be concluded that at 1 µM galantamine increases facilitation
of synaptic transmission by weakly, tonically activated nAChRs just
enough to override inhibition by strongly, tonically activated nAChRs.
Unoccupied, resting nAChRs may also exist in the Schaffer collaterals
(see Fig. 6). However, it is unlikely that galantamine-induced
potentiation of glutamatergic transmission is the result of direct
activation of these free receptors, because galantamine is a very weak
nAChR agonist (Pereira et al., 1994; Storch et al., 1995).
The bell-shaped concentration-response relationship for
galantamine-induced potentiation of glutamatergic transmission in hippocampal slices can be explained by the fact that up to 1 µM galantamine increases agonist-induced nAChR activation and
reduces/prevents agonist-induced nAChR desensitization, whereas at
higher concentrations, it inhibits agonist-induced nAChR activation
(Pereira et al., 1993; Schrattenholz et al., 1996; Samochocki et al.,
2000).
The effects of galantamine on field stimulation-evoked GABA-ergic
transmission in rat hippocampal slices and on ACh-triggered GABA
release in rat hippocampal and human cerebral cortical slices could
also be accounted for by its interaction with nAChRs located on
GABA-ergic neurons/axons synapsing onto the neurons from which recordings were obtained. The finding that galantamine alone did not
alter the frequency of spontaneously occurring IPSCs in rat hippocampal
slices ruled out a direct agonist action of the drug on presynaptically
located nAChRs that are in the resting, agonist-free state. Finally,
block by FK1 of galantamine-induced potentiation of ACh-triggered GABA
release in rat hippocampal and human cerebral cortical slices
demonstrated that the effect was the result of the nicotinic APL action
of galantamine.
Clinical Relevance of Galantamine Actions as a Nicotinic APL on
Synaptic Transmission in the Brain.
In the CNS, glutamatergic,
GABA-ergic, and cholinergic activities have been associated with
cognitive processing (Menschik and Finkel, 1998) and other forms of
synaptic plasticity, including dendritic spine motility and shaping
(Papa and Segal, 1996; Fischer et al., 2000; Shoop et al., 2001).
Cholinergic, glutamatergic, and GABA-ergic malfunctions have also been
associated with cognitive impairment in AD patients (Mohr et al., 1986;
Farber et al., 1998). Significant progress has been made in
understanding the role of nAChRs in the pathology of AD (Maelicke and
Albuquerque, 1996; Nordberg, 1999) and their relation to cognitive
function (Rezvani and Levin, 2001). Modulation of GABA-ergic and
glutamatergic transmissions by nAChRs seems to underlie the ability of
nicotinic agonists to improve learning and memory in animal models and
humans (Rezvani and Levin, 2001), and can be a major determinant of the
therapeutic effectiveness of galantamine in AD patients (Tariot et al.,
2000; Coyle and Kershaw, 2001).
; Tariot et al., 2000; Coyle and
Kershaw, 2001) and of rats in the step-down passive test (Bores et al.,
1996). There is a striking similarity between the dose dependence of
galantamine-induced enhancement of step-down passive avoidance in rats
(Bores et al., 1996) and the concentration dependence of
galantamine-induced facilitation of transmitter release (Fig. 2). In
both conditions, there is a bell-shaped relationship between
dose/concentration and effect. In addition, the brain concentration of
galantamine achieved after treatment of rats with the most behaviorally
active dose (2.5 mg/kg, p.o.) is ~0.8 µM (Bores et al., 1996). This
concentration is about the same as that at which galantamine exerted
its highest effect on synaptic transmission. This remarkable similarity
further underscores the relevance of modulation of transmitter release
to the reported memory-enhancing effects of galantamine in AD patients.
In conclusion, the present study provides evidence that at
concentrations that are behaviorally effective in enhancing cognitive tasks in animal models, galantamine, acting primarily as an APL at
presynaptically located nAChRs, facilitates glutamatergic and GABA-ergic transmissions in both rat and human brains. The development of drugs with nicotinic APL action represents a novel and effective approach to drug therapy for AD and other diseases linked to impaired nicotinic functions in the CNS.
| |
Acknowledgments |
|---|
We thank Mabel Zelle, Barbara Marrow, and Bhagavathy Alkondon for technical support.
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Footnotes |
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Received June 25, 2001; Accepted February 4, 2002
This work was supported by a grant from the Janssen Pharmaceutical Research Foundation, by United States Army Medical and Research Development Command Contract DAMD-17-95-C-5063, and by United States Public Health Service Grant NS25296 (to E.X.A.). A preliminary account of this study was presented at the 2000 Annual Meeting of the Society for Neurosciences (Soc Neurosci Abstr 26:1914, abstr. 716.5).
Address correspondence to: Dr. Edson X. Albuquerque, Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201. E-mail: ealbuque{at}umaryland.edu
| |
Abbreviations |
|---|
ACh, acetylcholine;
ACSF, artificial
cerebrospinal fluid;
AD, Alzheimer's disease;
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;
ANOVA, analysis
of variance;
APL, allosteric potentiating ligand;
APV, 2-amino-5-phosphonovaleric acid;
CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione;
CNS, central nervous system;
DHE, dihydro--erythroidine;
EPSC, excitatory postsynaptic
current;
GABA, -aminobutyric acid;
IPSC, inhibitory postsynaptic
current;
mIPSC, miniature inhibitory postsynaptic current;
MLA, methyllycaconitine;
nAChR, nicotinic acetylcholine receptor;
NMDA, N-methyl-D-aspartate;
PSC, postsynaptic
current.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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7-Nicotinic acetylcholine receptors and modulation of gabaergic synaptic transmission in the hippocampus.
Eur J Pharmacol
393:
59-67[CrossRef][Medline].42 subtype of neuronal nicotinic acetylcholine receptors stably expressed in fibroblast cells.
J Pharmacol Exp Ther
270:
768-77842 nAChR.
Acta Neurol Scand Suppl
176:
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|
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