1Neuroscience Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1; and 2Departments of Paediatrics, Anatomy, and Cell Biology, Clinical Trials Group, Canadian Paediatric Epilepsy Network, Queen's University, Kingston, Ontario K7L 3N6, Canada
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ABSTRACT |
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Fraser, Douglas D.,
Daniel Doll, and
Brian A. MacVicar.
Serine/Threonine Protein Phosphatases and Synaptic Inhibition
Regulate the Expression of Cholinergic-Dependent Plateau Potentials.
J. Neurophysiol. 85: 1197-1205, 2001.
We
previously identified cholinergic-dependent plateau potentials (PPs) in
CA1 pyramidal neurons that were intrinsically generated by interplay
between voltage-gated calcium entry and a
Ca2+-activated nonselective cation
conductance. In the present study, we examined both the
second-messenger pathway and the role of synaptic inhibition in the
expression of PPs. The stimulation of m1/m3 cholinergic receptor
subtypes and G-proteins were critical for activating PPs because
selective receptor antagonists (pirenzepine, hexahydro-sila-difenidol
hydrochloride, 4-diphenylacetoxy-N-methylpiperidine methiodide) and intracellular
guanosine-5'-O-(2-thiodiphosphate) prevented PP generation
in carbachol. Intense synaptic stimulation occasionally activated PPs
in the presence of oxytremorine M, a cholinergic agonist with
preference for m1/m3 receptors. PPs were consistently activated by
synaptic stimulation only when oxytremorine M was combined with
antagonists at both GABAA and GABAB receptors. These latter data indicate an
important role for synaptic inhibition in preventing PP generation.
Both intrinsically generated and synaptically activated PPs could not
be elicited following inhibition of serine/threonine protein
phosphatases by calyculin A, okadaic acid, or microcystin-L, suggesting
that muscarinic-induced dephosphorylation is necessary for PP
generation. PP genesis was also inhibited following irreversible
thiophosphorylation by intracellular perfusion with ATP--S. These
data indicate that the expression of cholinergic-dependent PPs requires
protein phosphatase-induced dephosphorylation via G-protein-linked
m1/m3 receptor(s). Moreover, synaptic inhibition via both
GABAA and GABAB receptors
normally prevents the synaptic activation of PPs. Understanding the
regulation of PPs should provide clues to the role of this regenerative
potential in both normal activity and pathophysiological processes such as epilepsy.
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INTRODUCTION |
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The cholinergic system has
been implicated in pathological activities such as epileptogenesis
(Lothman et al. 1991; Wasterlain et al.
1993
). Elevations in endogenous acetylcholine are associated with seizure onset (Mizuno and Kimura 1996
) and
cholinergic agonists facilitate epileptogenesis in kindled animals
(Buterbaugh et al. 1986
). In tissue slice preparations,
cholinergic receptor activation induces prolonged depolarizations
(Bianchi and Wong 1994
) and initiates ictal
depolarizations (Nagao et al. 1996
; Yaari and Jensen 1989
). We have previously identified a novel plateau
potential (PP) in hippocampal CA1 pyramidal neurons that has
characteristics similar to ictal depolarizations (Fraser and
MacVicar 1996a
). This regenerative PP was observed in the
presence of cholinergic (Haj-Dahmane and Andrade 1998
;
Klink and Alonso 1997
) or metabotropic glutamate
(Raggenbass et al. 1997
; Svirskis and Hounsgaard
1998
) agonists and requires interplay between calcium entry
through high-voltage-activated (HVA) Ca2+
channels and a Ca2+-activated nonselective cation
conductance (Congar et al. 1997
; Crepel et al.
1994
; Fraser and MacVicar 1996a
). Direct
enhancement of the Ca2+-activated nonselective
cation conductance by muscarinic receptor stimulation was suggested to
underlie PP expression; however, neither the muscarinic receptor
subtype(s) nor the signal transduction pathway have been identified.
Elucidating both the ionic mechanisms and the second-messenger pathways
underlying PPs could be essential for determining the role of this PP
in hippocampal epileptogenesis.
Muscarinic receptor activation modulates several voltage- and
ligand-gated ion channels (Halliwell 1990;
Krnjevic 1993
; McCormick 1993
) through a
number of second-messenger pathways. These include the phosphoinositide
cascade (Nicoll et al. 1990
), protein kinase C (PKC)
(Cantrell et al. 1996
; Figenschou et al.
1996
; Marsh et al. 1995
; Toselli and Lux
1989
; Zhang et al. 1992
),
Ca2+-calmodulin kinase II (CaMKII) (Muller
et al. 1992
; Pedarzani and Storm 1996
), tyrosine
kinase (Huang et al. 1993
), and serine/threonine protein
phosphastases (Herzig et al. 1995
; Krause and
Pedarzani 2000
). Protein kinases catalyze the transfer of a
phosphate from ATP to the side chain of an amino acyl residue,
resulting in structural changes of the target protein (i.e., ion
channels) (Hemmings et al. 1989
). The degree of ion
channel phosphorylation, however, depends not only on protein kinase
activity, but also on protein phosphatases that catalyze
dephosphorylation. Indeed, recent studies have demonstrated that the
phosphorylation of ion channels results from the dynamic equilibrium
between kinase and phosphatase activity (Bielefeldt and Jackson
1994
; Pedarzani et al. 1998
; Wang and Salter 1994
; Wilson et al. 1998
).
In the present study, we employed whole cell patch-clamp techniques in
the hippocampal slice preparation to investigate the putative receptor
subtype(s) and second messenger(s) underlying the expression of PPs in
CA1 pyramidal neurons. We also investigated the role of synaptic
inhibition on the synaptic activation of PPs. These results have been
presented in abstract form (Fraser and MacVicar 1996b).
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METHODS |
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Hippocampal slice preparation and whole cell patch-clamp recording
These techniques have been described previously (Fraser
and MacVicar 1996a). Sprague-Dawley rats, postnatal day
15-23, were decapitated and the brain immersed in chilled
artificial cerebrospinal fluid containing (ACSF; in mM): 126 NaCl, 2.5 KCl, 2 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO
Hippocampal slices were individually transferred to a recording chamber
located on an upright microscope (Standard 14; Zeiss, Thornwood, NY)
and submerged in rapidly flowing (1 ml/min) oxygenated ACSF
(34-35°C). Patch electrodes (5-7 M) were pulled from 1.5-mm OD
thin-walled glass (150F-4, World Precision Instruments) in two stages
on a Narishige puller (PP-83; Tokyo, Japan) and filled with
intracellular solution (in mM): 140 K-gluconate, 1.1 EGTA, 0.1 CaCl2, 10 HEPES, 2 Mg-ATP, and 0.3 Na-GTP, pH
7.2. Intracellular Ca2+ concentration was
calculated to be 16 nM. Voltage recordings were obtained in bridge mode
(Axoclamp-2A; Axon Instruments) and were low-pass filtered (4-pole
Bessel) at 10 kHz (
3 dB). Capacitance neutralization was fully
maximized, and series resistance was determined via a bridge circuit
potentiometer by balancing the voltage drop across the patch in
response to a negative current step (
30 pA; 10 ms). Data were
digitized via a Tl-1 A/D interface (Axon Instruments) and analyzed
using computer software (pCLAMP or Axotape). All data are presented as
means ± SE. To determine statistical significance, data groups
were prescreened for normality (Kolmogorov-Smirnov) and compared using
a Student's paired t-test (SigmaStat, Jandel Scientific).
Chemicals
All salts were purchased from Fisher (Fair Lawn, NJ), Sigma (St.
Louis, MO), or BDH (Toronto, ON). Carbachol (C-4832; Sigma), oxytremorine M (O-100; Sigma), atropine (A-0257; Sigma), pirenzipine (P-114; RBI; Natick, MA), 4-DAMP methiodide (D-104; RBI)
and Na-orthovanadate (S-6508; Sigma) were dissolved in distilled
H2O and added to the ACSF from concentrated
stocks. Also added to the ACSF, but first dissolved in DMSO, were
hexahydro-sila-difenidol hydrochloride (p-fluoro analogue;
H-127; RBI), calyculin A (C-3987; LC Laboratories; Woburn, MA), and
okadaic acid (O-2220; LC Laboratories). The final concentration of DMSO
was always 0.1%; in control experiments, DMSO at these
concentrations did not alter the cholinergic-dependent PP.
Second-messenger inhibitors and analogues that were dissolved directly
into the patch pipette solution included GDP-
-S (G-7637; Sigma), H-7
dihydrochloride (371955; Calbiochem; La Jolla, CA), ADP-
-S (A-8016;
Sigma), 2,4-dinitrophenol (D-7004; Sigma), alkaline phosphatase (P-1153; Sigma), ATP-
-S (A-1388; Sigma), and PP2B 476-501 (C-6481; LC Laboratories). Also added to the patch pipette solution, but first dissolved in DMSO (
0.1%) was microcystin-LR (M-173; RBI).
During all pharmacological manipulations, control experiments were alternated with drug experiments to ensure the presence of cholinergic-dependent PPs in untreated matched slices. In addition, it was imperative that the protein phosphatase inhibitors okadaic acid and microcystin-LR were made fresh immediately before use, as these compounds demonstrated reduced inhibitory activity when taken from frozen concentrated stocks.
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RESULTS |
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The results in this paper were obtained from 207 CA1 pyramidal
neurons in the hippocampal slice preparation. The whole cell patch-clamp method was used since this technique allows for both long-duration recordings and intracellular perfusion of
second-messenger inhibitors and analogues. The average access
resistance obtained in control neurons was 15.3 ± 0.4 M
(mean ± SE, range 7-20; n = 110); recordings
with series resistance >20 M
were discarded. The resting membrane
potential, input resistance, membrane time constant, and action
potential characteristics of the control cells were similar to our
previous reports (Table 1)
(Fraser and MacVicar 1996a
).
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Concentration dependence of the cholinergic-dependent PP
We investigated the activation of PPs in varying carbachol
concentrations. In all neurons tested under control conditions, depolarizing current injection (0.1 nA; 0.8 s) evoked repetitive action potential firing (Fig.
1A; n = 225),
and afterward the membrane potential immediately returned to the
prestimulation baseline. We tested the actions of different
concentrations of carbachol, a nonhydrolyzable cholinergic agonist, on
the afterpotentials following current-evoked action potential firing.
Bath application at each dose lasted for >5 min. In 0.1 µM
carbachol, as in control ACSF, the membrane potential returned to
prestimulation level following cessation of the current-evoked action
potentials (Fig. 1B; n = 6). However, in 0.3 µM carbachol, a slow afterdepolarization (sADP) of 4 ± 1 mV and
3.5 ± 0.4 s was observed immediately following the
depolarizing current stimulus (Fig. 1B; n = 6/6). A larger amplitude sADP was evoked when the carbachol
concentration was increased to 1 µM (7 ± 1 mV; 4.3 ± 0.4 s; n = 6/6). While action potential firing in
3 µM carbachol elicited a larger sADP in one-half the neurons
(14 ± 3 mV; 6.5 ± 0.4 s; n = 3/6), a
regenerative PP was observed in the remaining cells (n = 3/6). The membrane potential and duration of the PPs were
30 ± 8 mV and 2.8 ± 0.2 s, respectively. In the three
remaining neurons exhibiting only a sADP at 3 µM, PPs were observed
at a concentration of 10 µM. The membrane potential and duration of
the PPs in 10 µM carbachol were
20 ± 5 mV and 6.2 ± 0.9 s, respectively (n = 6/6). At the highest
concentration of carbachol tested (30 µM), PPs were elicited in all
neurons (n = 6/6). These data demonstrate a clear
concentration dependence of the cholinergic-dependent PP. A critical
level of receptor stimulation must be obtained between 3 and 10 µM
carbachol before a PP can be generated consistently. We therefore used
concentrations of 20 µM to elicit PPs so that we could examine drug
effects.
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Effects of muscarinic receptor antagonists
As reported previously, the PPs were abolished following
co-application of 1 µM atropine with 20 µM carbachol; although a small sADP was still elicited (2 ± 1 mV; 2.9 ± 0.5 s;
n = 5/5) (Fraser and MacVicar 1996a).
Since atropine is a nonselective antagonist of all muscarinic
receptors, we tested the ability of relatively selective muscarinic
receptor antagonists to suppress the PP elicited in the presence of 20 µM carbachol (Fig. 2). Coapplication of
either 1 µM pirenzipine (n = 3), an antagonist with
greater affinity for m1 over m3 receptors (Dorje et al.
1991
), or 1 µM 4-diphenylacetoxy-N-methylpiperidine methiodide
(n = 3; 4-DAMP), an antagonist with equal affinity to
both m1 and m3 receptors (Michel et al. 1989
;
Thomas et al. 1992
), abolished cholinergic-dependent PPs. Coapplication of 1 µM hexahydro-sila-difenidol hydrochloride (HHSiD; p-fluoro analog), an antagonist with greater
affinity for m3 over m1 receptors (Lambrecht et al.
1989
), also inhibited cholinergic-dependent PPs
(n = 3/4; data not shown). These data implicate m1
and/or m3 receptors in cholinergic-dependent PP genesis.
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G-protein involvement in the cholinergic-dependent PP
Muscarinic receptors are coupled to effector systems via
G-proteins (Brann et al. 1993), and some studies have
shown that G-proteins are involved in activation of hippocampal cation
currents (Crepel et al. 1994
). However, other recent
studies have excluded G-protein involvement in the muscarinic
activation of a nonselective cation conductance in cultured neurons
(Brown et al. 1993
; Guerineau et al.
1995
). To determine whether G-proteins are involved in the
generation of the cholinergic-dependent PP, we substituted 2 mM
guanosine-5'-O-(2-thiodiphosphate) (GDP-
-S) for GTP in
the patch pipette solution. GDP-
-S is a hydrolysis-resistant guanine nucleotide that inhibits receptor-induced activation of G-proteins (Andrade 1994
). Intracellular dialysis of hippocampal
CA1 pyramidal neurons with this compound abolished both the initial
membrane depolarization elicited by application of 20 µM carbachol
(data not shown; n = 3/3) and the cholinergic-dependent
PP (n = 3/3, Fig. 3).
These data indicate that G-proteins are a necessary component of the
cholinergic signal transduction pathway both in mediating the initial
depolarization and in the expression of the PP.
|
Role of dephosphorylation and protein phosphorylation
The regulation of ion channels by phosphatases has recently been
demonstrated (Bielefeldt and Jackson 1994; Wang
and Salter 1994
; White et al. 1991
), suggesting
that dephosphorylation may be important in the modulation of neuronal
responses. To test whether dephosphorylation plays a role in PP
genesis, ATP in the pipette solution was replaced with 5 mM
adenosine-5'-O-(3-thiotriphosphate) (ATP-
-S). We assumed
that there was some basal degree of protein kinase activity, and
therefore ATP-
-S was included in the patch pipette solution to
irreversibly thiophosphorylate substrate proteins. Neurons were
internally perfused with this compound for a minimum of 20 min prior to
data collection. Following this intracellular perfusion, the initial
depolarization induced by carbachol activation was significantly
reduced to 48 ± 14% of control (P
0.038, n = 6). Furthermore, expression of the
cholinergic-dependent PP was abolished (Fig.
4, A and B;
n = 6/6). These data indicate that dephosphorylation
was necessary for expression of cholinergic-dependent PPs.
|
We tested whether inhibitors of protein phosphatases could block the
expression of cholinergic PPs because the above data implicated that
dephosphorylation was necessary. Several approaches were used to test
for the involvement of serine/threonine protein phosphatases. First,
hippocampal slices (150-450 µm) were incubated in either calyculin A
(cal A) or okadaic acid (OA) at concentrations of 1-2 µM for >3 h.
This experimental approach has been used successfully in previous
studies, indicating that these inhibitors permeate neurons in tissue
slices (Mulkey et al. 1993; Muller et al.
1992
). Second, the cell-impermeable protein phosphatase
inhibitor microcystin-LR (m-LR), was applied internally via the patch
pipette at a concentration of 10 µM (Mulkey et al.
1993
). In this latter experiment, data collection began only
after 20 min of internal perfusion. The blockade of serine/threonine
protein phosphatases, by either calyculin A, okadaic acid, or
microcystin-LR, resulted in a depolarized resting membrane potential
(Table 1) and a reduction in the depolarization induced by 20 µM
carbachol (cal A, decreased to 33 ± 21% of control, P < 0.001, n = 7; OA, decreased to
44 ± 9% of control, P
0.003, n = 7; m-LR, decreased to 47 ± 25% of control,
P
0.008, n = 6). Finally, the
cholinergic-dependent PP could not be evoked in most neurons recorded
from slices incubated in either cal A (Fig. 4C;
n = 12/14 slices) or OA (Fig. 4D;
n = 6/7 slices). The cholinergic-dependent PP was also
abolished in 67% of neurons intracellularly loaded with m-LR (Fig.
4E; n = 4/6).
To examine the involvement of other protein phosphatase subtypes, we
also tested specific inhibitors of either protein phosphatase 2B
(calcineurin) or tyrosine phosphatase. To inhibit calcineurin, the
selective peptide inhibitor 476-501 (100 µM) (Hendey et al. 1992) was internally perfused via the patch-clamp electrode. At 100 µM, peptide 476-501 does not inhibit serine/threonine
protein phosphatases (Hendey et al. 1992
). Dialysis of
neurons for 20 min with this peptide inhibitor did not significantly
depress the initial cholinergic-induced depolarization (decreased to
90 ± 24% of control, n = 4) and was also
ineffective in inhibiting the cholinergic-dependent PP (data not shown,
n = 4/4). To test for the involvement of tyrosine
phosphatase, hippocampal slices were first incubated in 100 µM
Na-orthovanadate for >2 h, and this same concentration of inhibitor
was included in the patch pipette solution. At 100 µM,
Na-orthovanadate has virtually no dephosphorylating effect on
serine-threonine phosphoproteins (Swarup et al. 1982
).
This inhibitor neither affected the resting membrane potential nor
significantly depressed the initial cholinergic-induced depolarization
(decreased to 94 ± 16% of control, n = 4). In
addition, the cholinergic-dependent PP was consistently elicited (data
not shown, n = 4/4), suggesting that tyrosine
phosphatase is not a component of this signal transduction cascade.
Hence these data exclude the involvement of calcineurin or tyrosine
phosphatase in expression of the cholinergic-dependent PP.
Lack of effect of protein kinase inhibition
Our experiments with ATP--S and serine/threonine protein
phosphatase inhibitors indicated that dephosphorylation was critical for the expression of cholinergic-dependent PPs. Protein kinases are
reported to activate protein phosphatases directly in some signal
transduction pathways (Surmeier et al. 1995
;
Wilson and Kaczmarek 1993
). Therefore we tested whether
internal perfusion of 300 µM H-7, a potent, but nonselective kinase
inhibitor (Hidaka et al. 1991
; Ruegg and Burgess
1989
), could abolish PPs elicited in 20 µM carbachol
(Fraser et al. 1993
; Malenka et al. 1989
;
Malinow et al. 1989
; Zhang et al. 1992
).
The initial cholinergic-induced depolarization was significantly
reduced by H-7 in the patch pipette solution (decreased to 64 ± 8% of control; P
0.032, n = 4). In
contrast, H-7 did not block the expression of cholinergic-dependent PPs
elicited by action potential firing (Fig.
5A; n = 4/4).
These data imply that, although protein kinase(s) mediated some of the initial cholinergic-induced depolarization, there is no apparent involvement of protein kinases in expression of the
cholinergic-dependent PP.
|
We next determined the potential role for phosphorylation in the
expression of PPs elicited in 20 µM carbachol by internally perfusing
a phosphorylation-inhibiting cocktail (PIC) containing 0 Mg-ATP, 5 mM
ADP--S, 50 µM dinitrophenol, and 1 mg/ml alkaline phosphatase
(Chen and Smith 1992
; Chen et al. 1990
;
Hescheler et al. 1987
; Shuba et al.
1990
). The resting membrane potentials of neurons loaded with
PIC were significantly more depolarized than control neurons (PIC
59 ± 2 mV, n = 9; see Table 1 for control
values), and the initial cholinergic-induced depolarization was
significantly depressed in neurons containing PIC (decreased to 40 ± 14% of control, P
0.017, n = 4).
In contrast, the cholinergic-dependent PP was consistently elicited by
evoked action potential firing in the presence of 20 µM carbachol
(Fig. 5B; n = 4/4). This experiment further indicates that phosphorylation is unnecessary for the expression of cholinergic-dependent PPs.
Synaptic activation of PPs and GABA antagonists
The activation of PPs by current-evoked action potentials is an
all-or-none event (Fraser and MacVicar 1996a). We
investigated whether synaptic activation could also evoke all-or-none
PPs in CA1 pyramidal neurons by stimulating the Schaffer collaterals while bath applying a cholinergic agonist (carbachol or oxotremorine M). Oxotremorine M was sometimes used because it has slight preference for m1/m3 as compared with m2 cholinergic receptors (Brann et al. 1993
), and we showed above that m1/m3 receptors mediate PP genesis. In the presence of either carbachol (20 µM) or oxotremorine M (15 µM) alone, synaptic stimulation rarely activated PPs
(n = 2/6). In contrast, intracellular current injection
always evoked PPs in these same neurons (n = 6/6).
Synaptic stimulation did not evoke PPs even though the synaptic-induced
depolarization was equivalent to the depolarization elicited by
intracellular current injection that resulted in PP genesis (Fig.
6A). As inhibitory circuits
are also activated by synaptic stimulation, we perfused GABAergic
antagonists to determine whether synaptic stimulation would elicit PPs
when inhibition was depressed. Antagonists of GABAA receptors (30 µM bicuculline) and
GABAB receptors (50 µM CGP 35348) alone did not
unmask synaptically stimulated PPs (n = 0/6; Fig.
6B). Coapplication of oxytremorine M with either the GABAA antagonist bicuculline (30 µM;
n = 5), or with the GABAB receptor antagonist CGP 35348 (50 µM; n = 6) also did
not result in PP genesis with synaptic stimulation (data not shown).
However, PPs were consistently evoked under these conditions by
intracellular current injection (n = 8/11; oxytremorine
M, 15 µM; data not shown). In contrast, when both
GABAA and GABAB antagonists
(bicuculline 30 µM and CGP 35348 50 µM) were bath applied in
conjunction with oxotremorine M, synaptic stimulation consistently
evoked PPs (n = 21/24; Fig. 6C). These
results indicate that depression of both GABAA- and GABAB-mediated
synaptic inhibition and activation of muscarinic receptors
was necessary for the expression of synaptically driven PPs. In any one
cell, the waveform of the synaptically evoked PP was remarkably similar
to the PP evoked intrinsically by current injection (Fig.
6D). Similar to our previous report on intrinsically
generated PPs (Fraser and MacVicar 1996a
), PPs were not
evoked by synaptic stimulation in oxotremorine M, bicuculline, and CGP
34358 when 10 mM
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
was included in the patch pipette to chelate intracellular calcium
(n = 0/4; data not shown). Therefore synaptic
stimulation appears to evoke an intrinsically generated all-or-none PP
when muscarinic stimulation is combined with depression of synaptic inhibition.
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DISCUSSION |
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We have previously identified a novel cholinergic-dependent PP in
CA1 pyramidal neurons, which relies on the interaction between HVA
Ca2+ channels and the
Ca2+-activated nonselective cation conductance
(Fraser and MacVicar 1996a). This PP is observed in the
presence of cholinergic (or metabotropic glutamate) agonists and is
similar to ictal depolarizations observed during cholinergic-induced
seizures (Nagao et al. 1996
; Yaari and Jensen
1989
). In this paper, we have elucidated the muscarinic
receptors and various components of the signal transduction pathway
underlying the expression of cholinergic-dependent PPs. Our evidence
suggests that m1/m3 receptors are coupled to serine/threonine protein
phosphatases, either directly or indirectly via G-proteins. The
expression of cholinergic-dependent PPs therefore requires phosphatase-induced dephosphorylation. We have also demonstrated that,
similar to the generation of ictal depolarizations, the synaptic
activation of the PP is facilitated by depression of inhibition.
The expression of the cholinergic-dependent PP appears to be mediated
by m1 and/or m3 receptors. The presence of both m1/m3 receptor subtypes
in hippocampal tissue is well-documented by anatomical studies
(Vilaro et al. 1993), and m1/m3 receptor stimulation is
implicated in the modulation of cellular excitability (Auerbach and Segal 1996
; Cox et al. 1994
). The binding of
m1/m3 receptor antagonists, [3H]pirenzepine and
[3H]4-DAMP, is maximal in the rat hippocampus
relative to other structures (Aubert et al. 1996
), and
these antagonists inhibit phosphoinositide metabolism, suggesting that
stimulation of m1/m3 receptors liberate this second messenger
(Candura et al. 1995
). The phosphoinositide signal
transduction cascade activated by m1/m3 receptors is mediated by a
G-protein (Gq/11) (Vilaro et al.
1993
), and in agreement with this we have found that the
effects of cholinergic stimulation were occluded by GDP-
-S. Indeed,
several studies have demonstrated that muscarinic or metabotropic
glutamate receptor stimulation activates phospholipase C via
G-proteins, resulting in IP3 production and
elevated intracellular Ca2+ (Kostyuk and
Verkhratsky 1994
). Intracellular Ca2+
elevated by these neurotransmitters stimulates the
Ca2+-activated nonselective cation conductance,
thereby depolarizing the membrane potential (Congar et al.
1997
; Crepel et al. 1994
).
We have demonstrated critical roles for protein phosphatases and
dephosphorylation in the expression of cholinergic-dependent PPs. The
involvement of dephosphorylation was first tested using intracellular
perfusion of ATP--S, which irreversibly thiophosphorylates substrate
proteins. Intracellular dialysis of this compound depressed the initial
cholinergic-induced depolarization and abolished the PPs elicited in
carbachol. As dephosphorylation is mediated by protein phosphatases, we
then investigated whether inhibitors of protein phosphatases prevented
PP genesis. Both membrane-permeable (calyculin A and okadaic acid) and
-impermeable (m-LR) inhibitors of serine/threonine protein phosphatases
abolished the cholinergic-dependent PP. The effectiveness of these
inhibtors in our study is similar to a previous study of protein
phosphatases in hippocampal slices (Mulkey et al. 1993
).
Inhibitors of calcineurin (peptide 476-501) or tyrosine phosphatase
(Na+-orthovanadate) did not, however, affect PP
genesis. The expression of cholinergic-dependent PPs therefore required
the activation of serine/threonine protein phosphatases, either
directly or indirectly via G-proteins. A direct activation of protein
phosphatases by G-proteins has been suggested previously
(Bielefeldt and Jackson 1994
). Recently, muscarinic
modulation of calcium-activated potassium conductances has also been
shown to rely on protein phosphatase activation (Krause and
Pedarzani 2000
).
Protein phosphatases are directly activated by protein kinases in some
systems (Surmeier et al. 1995; Wilson and
Kaczmarek 1993
), and a variety of protein kinases are activated
by muscarinic stimulation (PKC, Figenschou et al. 1996
;
Marsh et al. 1995
; Zhang et al. 1992
;
CaMKII, Muller et al. 1992; Pedarzani and Storm
1996
; tyrosine kinase, Huang et al. 1993
).
Although the nonselective protein kinase inhibitor H-7 depressed the
initial cholinergic-induced depolarization in the present study, no
inhibition of the cholinergic-dependent PP was observed. This finding
suggested that PP genesis may be independent of protein kinase
activity. To further test this possibility, we internally perfused
neurons with PIC, a phosphorylation-inhibiting cocktail (Chen
and Smith 1992
; Chen et al. 1990
;
Hescheler et al. 1987
; Shuba et al.
1990
). Similar to the H-7 experiments, the initial
cholinergic-induced depolarization was depressed by PIC. The
cholinergic-dependent PP was still generated, however, again
demonstrating that phosphorylation is not involved in PP genesis.
What is the target for phosphatase-induced dephosphorylation? We have
previously demonstrated that the PP relies on interactions between
calcium entry through HVA Ca2+ channels and the
Ca2+-activated cation conductance (Fraser
and MacVicar 1996a). We further postulated that this latter
conductance is directly enhanced by cholinergic stimulation. Based on
the data presented here, we postulate that the target for
dephosphorylation is a serine/threonine site on the
Ca2+-activated nonselective cation channel. This
theory is consistent with previous reports demonstrating that the open
channel time and Ca2+ sensitivity of nonselective
cation channels are greatly reduced by PKA-induced phosphorylation in
both invertebrate (Partridge et al. 1990
) and vertebrate
neurons (Razani-Boroujerdi and Partridge 1993
).
Elevations in the activity of either cAMP or PKA also depress the
Ca2+-activated nonselective cation channel
recorded from cochlea (Van den Abbeele et al. 1996
) or
insulinoma (Reale et al. 1995
) cells. Consistent with
these findings, we have shown that the PP could not be generated in the
presence ATP-
-S or inhibitors of serine/threonine phophatases,
indicating the importance of dephosphorylation in PP genesis.
Interestingly, the conditions that reduce PP generation also enhance
activity of HVA Ca2+ channels. For example,
L-type Ca2+ currents in hippocampal neurons were
enhanced by elevations in intracellular PKA activity (Chetkovich
et al. 1991
; Hell et al. 1995
) or by protein
phosphatase inhibitors (Mironov and Lux 1991
). Given
that PPs rely on both HVA Ca2+ channels and the
Ca2+-activated nonselective cation conductance,
and that Ca2+ influx was probably augmented by
protein phosphatase inhibition, it is possible that the
Ca2+-activated nonselective cation conductance
was depressed to a much greater extent than is evident from our observations.
We have previously suggested that the PP is an excellent candidate for
an intrinsic mechanism underlying ictal depolarizations observed during
both experimental (Nagao et al. 1996; Yaari and Jensen 1989
) and clinical (Lothman et al. 1991
)
epileptogenesis. We have found that synaptic stimulation can elicit
PPs, but only when synaptic inhibition is depressed by GABA receptor
antagonists. Depression of synaptic inhibition is also an important
trigger for eliciting seizures in vivo (Lothman et al.
1991
). Our results support the hypothesis that the ictal
depolarization of epilepsy is an all-or-none PP that can be triggered
by spike activity or synaptic inputs (Fraser and MacVicar
1996a
). In addition, it has recently been demonstrated that a
clinically relevant anticonvulsant (10-100 µM topiramate)
depresses the cholinergic-dependent PP in subicular neurons
(Palmieri et al. 2000
). During interictal bursting,
elevated levels of ACh and glutamate could stimulate muscarinic and
metabotropic receptors, respectively. Activation of the protein
phosphatase pathway via these receptors, in conjunction with increased
intracellular calcium, may lead to PP genesis as described in this
study. Recurrent seizures can, as a consequence, induce profound
hypoglycemia (Wasterlain et al. 1993
) and precipitous decreases in ATP and phosphocreatinine levels (DeFrance and
McCandless 1991
; Fujikawa et al. 1988
). These
conditions would favor dephosphorylation and possibly exacerbate
existing seizure activity. As the PP represents a feed-forward
regenerative potential that results in prolonged depolarization and
maintained Ca2+ influx, these mechanisms may also
represent a crucial component of excitotoxicity (Chen et al.
1997
).
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ACKNOWLEDGMENTS |
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We are grateful to Drs. J. Armstrong, S. Kombian, S. Williams, and G. Spencer for comments on the manuscript.
This research was supported by grants from the Medical Research Council of Canada (MRC). D. D. Fraser received studentships from the MRC, the Alberta Heritage Foundation for Medical Research (AHFMR), and the Savoy Foundation for Epilepsy Research. D. Doll was supported by an MRC studentship. B. A. MacVicar is an AHFMR scientist and an MRC Senior Scientist.
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FOOTNOTES |
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Address for reprint requests: B. MacVicar, Dept. of Physiology and Biophysics, 3330 Hospital Drive N.W., University of Calgary, Alberta T2N 4N1, Canada (E-mail: macvicar{at}ucalgary.ca).
Received 10 July 2000; accepted in final form 22 November 2000.
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REFERENCES |
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