Departments of 1Neuroscience and 2Neurology, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Santschi, Linda, Magali Reyes-Harde, and Patric K. Stanton. Chemically Induced, Activity-Independent LTD Elicited by Simultaneous Activation of PKG and Inhibition of PKA. J. Neurophysiol. 82: 1577-1589, 1999. Although it is widely agreed that cyclic AMP is necessary for the full expression of long-term potentiation of synaptic strength, it is unclear whether cyclic AMP or cyclic AMP-dependent protein kinase (PKA) play roles in the induction of long-term depression (LTD). We show here that two PKA inhibitors, H-89 (10 µM) and KT5720 (1 µM), are unable to block induction of LTD at Schaffer collateral-CA1 synapses in hippocampal slices in vitro. Rather, H-89 enhanced the magnitude of LTD induced by submaximal low-frequency stimulation. Raising [cGMP] with zaprinast (20 µM), a selective type V phosphodiesterase inhibitor, reversibly depressed synaptic potentials. However, coapplication of H-89 plus zaprinast converted this to a robust LTD that depended critically on activation of cyclic GMP-dependent protein kinase (PKG). Chemically induced LTD is activity-independent because it could be induced without stimulation and in tetrodotoxin (0.5 µM). Additionally, chemical LTD did not require activation of N-methyl-D-aspartate or GABA receptors and could be reversed by LTP. Stimulus-induced LTD occluded chemical LTD, suggesting a common expression mechanism. In contrast to bath application, postsynaptic infusion of H-89 into CA1 pyramidal neurons did not enhance LTD, suggesting a presynaptic site of action. Further evidence for a presynaptic locus was supplied by experiments where H-89 applied postsynaptically along with bath application of zaprinast was unable to produce chemical LTD. Thus simultaneous presynaptic generation of cyclic GMP and inhibition of PKA is sufficient to induce LTD of synaptic transmission at Schaffer collateral-CA1 synapses.
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INTRODUCTION |
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Homosynaptic long-term depression (LTD) is an
input-specific, long-lasting reduction in synaptic strength that can be
generated by prolonged low-frequency stimulation in a number of
cortical areas, including at Schaffer collateral/commissural-CA1
synapses in the hippocampus (for review, see Bear and Abraham
1996; Christie 1996
). Although the precise,
physiologically relevant functions of LTD are still unclear, most
neural network models include both long-term increases and decreases in
synaptic strength to accomplish memory storage, information processing,
and mnemonic classifications (Bienenstock et al. 1982
;
Sejnowski 1977
). It is a popular hypothesis that
memories may have their cellular correlate in precise spatiotemporal patterns of synaptic strengths. Since the demonstration of
homosynaptic, associative LTD (Stanton and Sejnowski
1989
), much work has focused on the elucidation of cellular
mechanisms underlying both the induction and expression of cortical LTD.
Cyclic nucleotide signal transduction cascades have been
suggested to play a role in synaptic plasticity in a wide variety of
systems. In mammalian hippocampus, it is now well established that
cyclic AMP-activated pathways play necessary roles in the full
expression of long-term potentiation (LTP) of synaptic strength (Abel et al. 1997; Blitzer et al. 1995
;
Frey et al. 1993
; Hopkins and Johnston
1988
; Matthies and Reymann 1993
; Stanton
and Sarvey 1985a
,b
; Stanton et al. 1989
).
Although most of these studies have focused on a late-phase of LTP
involving both activation of postsynaptic kinases and alterations in
gene expression and protein synthesis, there is also evidence that
supports a potential presynaptic role of cyclic AMP at mossy fiber
terminals in field CA3 (Huang et al. 1994
) and at
Schaffer collateral/commissural terminals in field CA1 of rat
hippocampal slices (Chavez-Noriega and Stevens 1994
).
Recent work in our and other laboratories (Gage et al.
1997; Izumi and Zorumski 1993
;
Reyes-Harde et al. 1999a
,b
) has linked another cyclic
nucleotide second-messenger cascade, the nitric oxide (NO)-cyclic GMP
system, to the induction of LTD. As a first step toward identifying
downstream effectors of cyclic GMP, recent evidence (Reyes-Harde
et al. 1999a
,b
) implicates presynaptic cyclic GMP-dependent
protein kinase (PKG) as a necessary component in the LTD induction
pathway. Thus cyclic AMP and cyclic GMP appear to be involved
reciprocally in long-term regulation of synaptic strength. However, it
also has been reported that cyclic AMP-dependent protein kinase (PKA)
activity is necessary to permit the induction of LTD (Brandon et
al. 1995
). Given that PKA and PKG have reciprocal actions in
many cells, (Inoue et al. 1995
;
Polanowska-Grabowska and Gear 1994
; Wexler et al.
1998
), it seemed surprising to us that PKA activation would be
necessary for both LTP and LTD. We set out to test our hypothesis of a
bidirectional regulation model in which these two cyclic nucleotides
would have opposing actions on LTD. We show here that inhibition of PKA
results in an enhancement of stimulus-evoked LTD via a PKG-dependent
mechanism. In addition, we demonstrate that blockade of the hydrolysis
of cyclic GMP, in conjunction with simultaneous inhibition of PKA,
elicits a sustained, chemically induced, activity-independent LTD
that appears to share mechanisms in common with stimulus-evoked LTD.
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METHODS |
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Transverse hippocampal slices (400-µm thick) were obtained
from Sprague-Dawley rats of either sex (14-21 days old). Briefly, animals were anesthetized and decapitated. The brain was removed rapidly, the hippocampus dissected and placed in 4°C artificial cerebrospinal fluid (ACSF; bubbled with
95%O2-5%CO2, pH 7.4), which comprised in (mM) 126 NaCl, 26 NaHCO3, 1.25 NaH2PO4, 5 KCl, 2 CaCl2, 2 MgCl2, and 10 D-glucose. Slices were cut, using an "egg-slicer,"
which rapidly forced a parallel grid of 20-µm-diam wires spaced 400 µm apart through the tissue. Individual slices were placed in an
interface recording chamber at 33°C, and perfused at a rate of 3 ml/min with ACSF. When appropriate, one input served as a control to
verify the input specificity of LTD. Evoked population excitatory
postsynaptic potentials (fEPSPs) were recorded extracellularly in the
apical dendritic field in stratum radiatum for a stable baseline period of 30 min. Stimulation intensity was adjusted so that
the amplitude of each fEPSP was ~50% of the maximum response amplitude before generation of a population action potential (
2 mV).
Experiments in which there was a drift in baseline of >5% were
excluded from further analysis. De novo LTD was induced by a 1 Hz/15
min (900 stimuli) train of low-frequency stimulation (LFS). A
"submaximal" LFS consisted of 400 pulses, also at a frequency of 1 Hz. For the reversal experiments, LTP was induced by a theta burst
stimulation (TBS) paradigm where we gave 10 high-frequency bursts (100 Hz, 5 pulses each) repeated at 5 bursts/s (200-ms interburst interval).
This TBS was repeated four times spaced 15 s apart. The stimulus
intensity was increased during TBS so as to double the amplitude of the
evoked EPSP. In experiments performed in the presence of GABAergic
antagonists, [Mg2+]o was
raised to 4 mM, and area CA1 was isolated by making a cut in stratum
radiatum between fields CA3 and CA1. For the occlusion experiments, two
separate inputs of Schaffer collateral-commissural axons were isolated
by placement of stimulating electrodes on opposite sides of the
recording site (extracellular electrodes filled with 2 M NaCl),
verified as separate inputs by a lack of paired-pulse interactions
between them (50 ms interstimulus intervals), and alternately
stimulated every 30 s using bipolar, stainless steel electrodes
(150µs DC square pulses). Synaptic strength was assessed by measuring
the maximum slope of the falling phase of the fEPSP. Changes in
synaptic strength after LFS and/or drug addition are expressed relative
to the normalized pre-LFS baseline. The maximum initial negative slopes
of the fEPSPs were calculated using a six-point interpolation
least-squares linear regression analysis. Summary data are expressed as
mean ± SE with significance preset to the P < 0.05 level, using Student's t-tests for unpaired data.
We recorded intracellular evoked EPSPs in CA1 pyramidal neurons
(RMP = 61 ± 1.6 mV; RN = 55.8 ± 2.4 M
) impaled with sharp microelectrodes (90-130
M
, 2 M KAcetate). Maximum initial positive slope of the EPSP (V/s)
was measured to assess changes in excitatory synaptic strength, and
each experiment normalized to its pre-LFS baseline value. In most
experiments, simultaneous field recordings were made to monitor
recording condition stability. Stock solutions of H-89 (Biomol), KT5720
(LC Labs/Alexis Corp), KT5823 (Biomol), picrotoxin (Sigma), and
zaprinast (Sigma) were dissolved in dimethyl sulfoxide (DMSO) as
1,000× stocks and diluted either in ACSF for bath application or in 2 M KAcetate for intracellular infusion. D-2-amino-5-phosphonopentanoic acid (D-AP5; Tocris Cookson)
was dissolved in aqueous NaOH. N
-L-nitro-arginine
(Sigma) was dissolved in 1 M HCl. Tetrodotoxin (Sigma) and CGP
35348 (kind gift of Ciba-Geigy Pharmaceuticals) were dissolved in
dH2O to a stock concentration of 1 mM and 0.5 M,
respectively. Forskolin (Sigma) was dissolved in DMSO to a 50 mM stock
concentration. Intracellular and extracellular control experiments used
equal concentrations of DMSO vehicle alone, and expression of LTD was
unaffected by this vehicle at any concentration employed.
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RESULTS |
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PKA inhibitors do not block the induction of homosynaptic LTD
To test the necessity for PKA activation for the induction of LTD
at Schaffer collateral-CA1 synapses, we employed two selective, cell-permeant PKA inhibitors, H-89 (a synthetic inhibitor)
(Chijiwa et al. 1990) and KT5720 (a secondary
metabolite-derived inhibitor) (Kase et al. 1987
). Figure
1A illustrates the induction of
LTD by LFS (1 Hz/900 s; solid bar) in the presence of bath-applied H-89
(10 µM; open bar). LTD in these slices was indistinguishable from
control LTD (
36.6 ± 2.9%; n = 16, data not
shown) in either magnitude or duration (LTD in H-89 =
35.1 ± 6.0%; P > 0.20; Student's t-test
compared with controls; n = 9). Similarly, the second
PKA inhibitor, KT5720 (1 µM; open bar; Fig. 1B), was also
unable to block the induction of LTD by LFS (LTD in KT5720 =
35.2 ± 6.4%; n = 6). Taken together, these
experiments lead to the conclusion that PKA activation is not a
necessary step in the induction of homosynaptic LTD at Schaffer
collateral-CA1 synapses.
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Homosynaptic LTD is enhanced by inhibiting PKA
The preceding observations, and previous studies showing that PKA
activity is necessary for the full expression of LTP (Frey et
al. 1993; Matthies and Reymann 1993
;
Stanton and Sarvey 1985a
,b
; Stanton et al.
1989
), raise the question of whether preventing activation of
PKA in fact might enhance or unmask the induction of a reciprocal
modification of synaptic strength, LTD. To test this hypothesis, we
used a shorter, submaximal LFS stimulation, 1 Hz for 400 s, which
induced only a small amount of LTD in control slices (Fig.
1C;
11.5 ± 2.9%; n = 11). In a
second group of slices (Fig. 1D; n = 14), we
bath-applied H-89 (10 µM; open bar) 45 min before submaximal LFS of
Schaffer collateral/commissural axons (1 Hz/400 s; solid bar). In
contrast to control slices, submaximal LFS elicited virtually maximal
LTD when PKA was inhibited (
35.4 ± 4.1%, P < 0.05; Student's t-test compared with controls 60 min post-LFS). This intriguing result prompted further investigation into
the synaptic locus of action of H-89 as well as studies aimed at
testing the possibility of a co-requirement for an NO-cyclic GMP-PKG
pathway (Gage et al. 1997
) in this "unmasked" LTD.
Postsynaptic PKA inhibition does not enhance LTD
In a recent study (Gage et al. 1997), we presented
evidence that at least one form of homosynaptic LTD requires activation of a presynaptic cyclic GMP-mediated cascade triggered by the gaseous
intercellular messenger NO. To test the possibility that PKA
counteracts this cascade directly, it was necessary to determine whether H-89 acts pre- and/or postsynaptically. To accomplish this, we
used sharp intracellular microelectrodes to fill single CA1 pyramidal
neurons with H-89. Intracellular electrodes were backfilled with a
solution containing a 50-fold higher concentration of H-89 (500 µM)
than that which was effective in enhancing LTD when applied extracellularly.
Figure 2A illustrates the effect
of submaximal Schaffer collateral LFS (1 Hz/400 s; solid bar) on
intracellularly recorded EPSPs in control, untreated CA1 pyramidal
neurons (n = 7). The left inset shows
examples of single EPSPs before and after submaximal LFS in a control
cell. The right inset illustrates burst-induced afterhyperpolarization (AHPs; +0.5-1.0nA/100 ms injected current) before and after bath application of the adenylate cyclase stimulant forskolin (100 µM), which elicited a well-characterized PKA-mediated block of the late AHP (Madison and Nicoll 1986).
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Figure 2B illustrates the effects of identical submaximal
LFS on CA1 pyramidal neurons filled with H-89 to selectively inhibit postsynaptic PKA. CA1 pyramidal neurons were impaled with
microelectrodes filled with 500 µM H-89 (in 2 M
K+ acetate + 0.5% DMSO; Re = 90-130 M). At least 45 min were allowed for leakage of H-89 into
neurons (open bar), after which submaximal LFS (1 Hz/400 s; solid bar)
was applied to one Schaffer collateral input, while a second input
served as control (not shown). In contrast to extracellular application
of H-89, selective inhibition of postsynaptic PKA did not statistically
enhance the magnitude of LTD evoked by submaximal LFS (LTD in
control =
10.4 ± 6.7%, H-89 treated =
17.4 ± 4.3%, n = 8; P > 0.20, Student's
t-test compared with control intracellular EPSPs 30 min
post-LFS). The left inset illustrates representative EPSPs before and
after submaximal stimulation.
To verify that postsynaptic PKA in fact was blocked under our recording
conditions, we tested the ability of the adenylate cyclase stimulant
forskolin to block the burst-evoked AHP in three of these cells. The
right inset illustrates substantial but not complete
prevention of forskolin's actions in cells infused with H-89 compared
with control cells, confirming blockade of PKA. In fact, H-89 (10 µM)
produced virtually identical (80%) block of forskolin's actions
when applied extracellularly or intracellularly. Taken together, these
experiments suggest that the majority (but perhaps not all) of the
enhancement of LTD produced by inhibition of PKA is most likely due to
a presynaptic site of action.
NO synthase is necessary for the form of LTD unmasked by inhibiting PKA
We have shown recently that full LFS-evoked homosynaptic LTD
consists of both NO-guanylyl cyclase-dependent and -independent components (Gage et al. 1997). However, it was not clear
which component(s) contribute to the LTD unmasked by inhibiting PKA in
our current experiment. Therefore, we used the cell-permeant NO
synthase inhibitor N
-nitro-L-arginine (L-NA) to determine whether NO production is, in fact, a necessary component of LTD evoked
under either condition. Figure 3A
(n = 6) illustrates that, in agreement with previous
reports (Izumi and Zorumski 1993
; Otani and
Connor 1995
), bath application of L-NA (100 µM; open bar) partially reduced the magnitude of LTD produced by LFS (1 Hz/900 s;
solid bar;
23.7 ± 2.9% compared with
36.6 ± 2.9% in
controls). NOS inhibition did not, however, completely block LTD,
consistent with the existence of an NO-independent component of LTD
(Gage et al. 1997
).
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Next, we coapplied L-NA (100 µM) plus H-89 (10 µM) 45 min before
giving submaximal LFS (1 Hz/400 s; solid bar). Figure 3B (n = 8) illustrates that the robust LTD previously seen
in the presence of H-89 was blocked almost completely by inhibition of NOS activity. The existence of a small, residual amount of depression (17.4 ± 5.5%) suggests that a part of the observed H-89 effect also may be NO-independent, perhaps acting through a postsynaptic cascade involving protein phosphatases (Mulkey et al.
1993
).
Cyclic GMP accumulation alone is not sufficient to induce LTD
One physiological function of presynaptic nitric oxide is to bind
to the heme moiety of soluble guanylyl cyclase (sGC), causing the
synthesis of cGMP. However, it was unclear whether an elevation in
[cGMP] alone could be sufficient to elicit LTD. Isozyme-specific phosphodiesterase (PDE) inhibitors are useful pharmacological tools for
examining the physiological roles of cyclic nucleotides in a variety of
systems (Beavo 1995). In particular, it is the type V
PDE that is primarily responsible for the selective breakdown of cyclic
GMP. Therefore, a selective PDE V inhibitor, such as zaprinast,
markedly and selectively elevates intracellular [cGMP] (Gillespie and Beavo 1989
).
Figure 4A shows that bath
application of zaprinast (5 µM; open bar; n = 6)
caused a transient depression of synaptic EPSPs that fully reversed
within 30 min of drug washout. Thus raising [cGMP] by this method, in
the absence of any stimulus train, produced only a transient, rapidly
reversible short-term depression, consistent with previous reports
(Boulton et al.1994). Similarly, Fig. 4B demonstrates that zaprinast (open bar; n = 6), added
along with coincident submaximal LFS (1 Hz/400 s; solid bar), produced
a depression that, though much larger in magnitude, was still fully reversible on drug washout. Using a higher concentration of zaprinast (20 µM), Fig. 4C (n = 8) shows that, even
at this concentration, zaprinast-induced depression was still rapidly
and completely reversible on drug washout. Thus, the reversibility of
zaprinast's effect is probably not attributable to some threshold
level in the [cGMP] achieved. Zaprinast at this concentration (20 µM), paired with submaximal stimulation, also produced only a
reversible depression (n = 2, data not shown). Taken
together, these experiments indicate that simply elevating [cGMP]
alone is insufficient to elicit fully saturated, stable LTD of synaptic
strength.
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Simultaneously raising [cyclic GMP] while inhibiting PKA induces LTD
Studies in our and other laboratories (Boulton et al.
1994; Gage et al. 1997
) support a role for cGMP
in synaptic depression at CA1 synapses in rat hippocampal slices.
Because raising cGMP alone is insufficient to produce a long-lasting
depression, but we do find a marked enhancement in the magnitude of LTD
when another signaling system (PKA) is inhibited, we hypothesized that
these two pharmacological manipulations in concert might supply the factors needed to produce a long-lasting depression of synaptic strength.
In contrast to the effect of zaprinast alone, Fig. 4D
illustrates the induction of an apparently saturated LTD when zaprinast (20 µM; solid bar; n = 8) and H-89 (10 µM; open
bar) were coapplied. This "chemical LTD" remained stable for 2 h
following washout of both drugs (
41.8 ± 3.8%,
P < 0.05, Student's t-test, 60 min postapplication compared with preapplication baselines). These data
support our hypothesis that these two cyclic nucleotide second messengers mediate opposing biological signals and that simultaneous elevation of [cGMP] and inhibition of PKA is, in fact,
sufficient to elicit LTD at Schaffer collateral-CA1 synapses.
Chemical LTD is not associated with alteration in paired-pulse facilitation
Our intracellular data with postsynaptic infusion of H-89
(Fig. 2B) suggests that the synaptic locus of this effect
may be presynaptic. A common, but not universal, feature of a
presynaptic site of action is an alteration in paired pulse
facilitation (PPF) (Zucker 1989). PPF is an enhancement
in the response magnitude to a second stimulus when preceded at a short
interstimulus interval (10-100 ms) by a conditioning stimulus. PPF is
believed, though not without debate, to result from residual-free
Ca2+ in the presynaptic terminal, which is
retained near sites of transmitter release for a period of time after
the conditioning pulse (Katz and Miledi 1968
; but see also
Bertram et al. 1996
).
We compared PPF ratios before and after the induction of chemical LTD (Fig. 5). The pooled chemical LTD that was elicited is illustrated in Fig. 5A (n = 13) and was similar to previous experiments. Associated PPF was measured at two interstimulus intervals, 50 ms (Fig. 5B; n = 8) and 15 ms (Fig. 5C; n = 5). PPF at either paired-pulse interval was not consistently altered during chemical LTD.
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Chemical LTD requires PKG activity
While cGMP can exert its biological effects through a number of
effector cascades, it is unclear which of these downstream pathways are
required to induce LTD. A primary action of cGMP is the stimulation of
PKG, which in many systems is considered the primary intracellular
receptor protein for cGMP. Recent data from our laboratory
(Reyes and Stanton 1997) demonstrate that PKG activation
is necessary for the induction of LTD. This result, together with our
present data involving PKA inhibition, led us to hypothesize a role for
reciprocal regulation of long-term synaptic plasticity by the two
cyclic nucleotide-activated protein kinases. To determine if the LTD
observed in H-89 also requires the coincident activation of PKG, we
used the selective, cell-permeant PKG inhibitor KT5823. Figure
6A illustrates that, when H-89 and
KT5823 (10 and 1 µM respectively; open bar) were co-bath applied 45 min before submaximal LFS (1 Hz/400 s; solid bar), the H-89 unmasking
of LTD was blocked completely (
4.3 ± 1.9%; n = 10). This shows that the unmasking of LTD by inhibiting PKA does
require the unopposed activity of PKG. Just as significantly, this
result also ensured that the LTD-enhancing effect seen with H-89 was,
in fact, due exclusively to PKA inhibition and not attributable to
cross-inhibition of PKG.
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Because LTD in H-89 was prevented by simultaneous addition of KT5823,
we investigated whether PKG activity also is needed for chemically
induced LTD. Figure 6B illustrates that blockade of PKG
could, indeed, prevent the expression of chemical LTD produced by
raising [cGMP] while inhibiting PKA (60 min postwash; 6.1 ± 9.7%; n = 4). These data lend strong support to the
contention that PKG is the primary physiological target for cyclic GMP
generated in response to zaprinast and that PKG activity unopposed by
PKA is what is required to yield stable, long-lasting LTD.
Both PKG and PKA function presynaptically to regulate chemical LTD
Before considering possible substrates for PKG, it was first
necessary to determine the synaptic locus of PKG activity required for
LTD. To address this, we dissolved KT5823 (50 µM) at 50 times the
extracellularly effective concentration in 2 M potassium acetate plus
0.5% DMSO and backfilled microelectrodes for intracellular perfusion.
Intracellular evoked EPSPs in single CA1 pyramidal neurons were
recorded for a baseline period of 45 min to allow KT5823 to leak into
the postsynaptic neuron. H-89 (10 µM) then was bath applied alone for
10 min, followed by 30-min bath application of H-89 plus zaprinast (20 µM). Figure 6C illustrates the results of these
experiments in which postsynaptic infusion of KT5823 was unable to
block the expression of chemical LTD (
43.1 ± 1.2%; n = 6). This result supports the hypothesis that
presynaptic, not postsynaptic, PKG is necessary for chemical LTD.
Our previous experiment (Fig. 2) suggests a presynaptic locus for PKA
inhibition in the enhancement of LTD; however, the site of action of
H-89 in the expression of chemical LTD could be different. To answer
this question, we backfilled electrodes with H-89 (500 µM) for
intracellular infusion of CA1 pyramidal neurons. After 45 min of
infusion and baseline recording, we bath applied zaprinast (20 µM)
for 30 min, followed by drug washout. Figure 6D shows that
chemical LTD could no longer be elicited when H-89 was applied postsynaptically (10.5 ± 8.5%; n = 6),
indicating that the negative role for PKA important to the induction of
LTD is probably also presynaptic.
Chemical LTD is reversed by HFS and occluded by stimulus-evoked LTD, but is activity-independent
Associated with studies of LTD is an ongoing concern that
depression of synaptic potentials may reflect irreversible synaptic damage rather than a reversible physiological phenomenon. Figure 7A demonstrates that, similar to
stimulus-induced LTD, chemical LTD can be reversed by the induction of
LTP. After inducing stable chemical LTD (33.3 ± 0.76%),
high-frequency stimulation (HFS; theta burst stimulation; 4 trains of
100 Hz/5 pulse burst times 10, interburst interval 200 ms; see
METHODS ) was applied 45 min after drug washout (TBS
indicated by arrows), which evoked robust LTP (+208% change from
chemically depressed baseline). fEPSPs remained stably potentiated for
1 h, at which time a second TBS was given which resulted in a
slight additional potentiation. A third TBS given 45 min later elicited
no further potentiation, indicating that LTP was saturated at this
level. This demonstration of reversibility argues against irreversible
damage underlying chemical LTD.
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These data also hint that stimulus-induced and chemical LTD may share
some common mechanisms because both can be reversed by the same HFS. We
set out to test the hypothesis that these may not be separate and
distinct forms of LTD by determining whether saturation of
stimulus-induced LTD at one set of synapses could selectively occlude
chemical LTD. As shown in Fig. 7B, we alternately stimulated
each of two independent Schaffer collateral pathways once every 30 s (S1 and S2) and recorded the evoked fEPSPs in stratum radiatum of
field CA1. After the baseline period, the S1 input (solid circles)
received repeated LFS trains (4-5 trains, 1 Hz/600 s), spaced 10 min
apart, until saturated, homosynaptic LTD was induced. Ten minutes after
the last train, we bath applied H-89 (10 µM; open bar) for 30 min,
followed by coapplication of H-89 plus zaprinast (20 µM; solid bar)
for an additional 30 min. In the presence of both inhibitors, both
inputs did, indeed, decrease markedly in strength. However, on drug
removal, the saturated input (S1) returned to its former level of
depression (S1 saturated, prechemical LTD: 32.8 ± 4.7%;
postchemical LTD:
36.2 ± 5.6%). In contrast, the naive input
(S2, open circles) demonstrated chemical LTD that was indistinguishable
in magnitude from stimulus-induced LTD (
42.0 ± 6.4%,
P > 0.20, Student's t-test comparing S1 to S2). The occlusion of chemical LTD by stimulus-evoked LTD further supports the hypothesis that they share at least some common expression mechanisms.
To determine whether chemical LTD requires any synaptic activity
at all for its generation, we performed experiments where all synaptic
stimulation was suspended during the period of drug application and not
resumed until 30 min postwashout. As shown in Fig. 7C,
virtually identical chemical LTD was elicited in the absence of
electrical stimulation (37.1 ± 3.1%, n = 5).
To completely silence both evoked and spontaneous synaptic activity, we
also performed the same experiment in the presence of the
Na+ channel blocker TTX (0.5 µM). As Fig.
7D shows, chemical LTD was induced in the presence of TTX,
after which all three drugs were washed out. Although considerable time
was needed for TTX to completely wash out of these slices (
80 min),
once fEPSPs stabilized, the magnitude of LTD was the same as in
controls (
36.2 ± 4.3%; n = 4). Thus,
chemically induced LTD does appear to converge on some of the same
mechanisms as stimulus-induced LTD while bypassing the need for any
synaptic stimulation.
Induction of chemical LTD does not require either NMDA or GABA receptor activation
An initial trigger for one form of stimulus-induced LTD is
activation of the N-methyl-D-aspartate (NMDA)
class of glutamate receptors (Dudek and Bear 1992). One
possible physiological cascade for raising presynaptic [cGMP] begins
with the activation of postsynaptic NMDA receptors,
Ca2+ influx, and activation of the
Ca2+/calmodulin-dependent enzyme NO synthase
(NOS). NO then diffuses readily across membranes, allowing it to act on
neighboring cells and/or presynaptic terminals (Boulton et
al.1994
). One of the targets activated by NO is soluble
guanylate cyclase (sGC), elevating [cGMP]. It seems likely that
chemical LTD, by directly elevating [cGMP], bypasses the need for
NMDA receptor activation. To test whether NMDA receptor activation is
required for chemical LTD, we attempted to elicit chemical LTD in the
presence of the NMDA receptor blocker D-AP5. Figure
8A shows that chemical LTD still could be evoked despite NMDA receptor blockade (50 µM AP5; hatched bar; n = 8). These results demonstrate that effectively
raising [cGMP] in response to zaprinast is enough to overcome the
need for NMDA receptor-gated Ca2+ influx,
provided an opposing cAMP-activated signaling pathway is inhibited
concomitantly.
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In considering the synaptic locus of chemical LTD, alterations in
inhibitory circuitry that modulate excitatory transmission are also
potential contributors, even though blockade of GABAergic inhibition
has been shown rather to enhance stimulus-evoked LTD (Wagner and
Alger 1995). Trudeau et al. (1997)
have shown
that hippocampal inhibitory postsynaptic potentials (IPSPs) are
unusually sensitive to PKA activation, raising the possibility that the effect of PKA inhibition could include an indirect effect on inhibitory tone. Therefore, we tested whether chemical LTD still could be elicited
under conditions of GABA receptor blockade. Experiments were conducted
in the presence of both the GABAA receptor
antagonist picrotoxin (10 µM), and the GABAB
receptor blocker, CGP 35348 (400 µM; hatched bar). Figure
8B (n = 8) shows that the expression of
chemically induced LTD does not require intact GABAergic modulation, supporting a direct effect of chemical LTD on monosynaptic excitatory transmission.
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DISCUSSION |
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While there has been some progress in elucidating cellular mechanisms underlying LTD, we are far from a complete understanding of either the molecular processes or cognitive function(s) of this form of plasticity. Interestingly, many of the same second-messenger cascades appear to be involved in both LTP and LTD, leading one to question where the critical points of divergence in the two phenomena may be. Here, we supply the first evidence of reciprocal regulation of LTD by cyclic GMP-(LTD enhancing) and cyclic AMP-(LTD suppressing) dependent protein kinases in presynaptic nerve terminals.
The fact that PKA plays an important role in the persistence of LTP is
well established (Frey et al. 1993; Hopkins and
Johnston 1988
; Stanton and Sarvey 1985a
,b
;
Stanton et al. 1989
). If PKA-mediated phosphorylation of
protein substrates favors the expression of LTP, we predicted that
inhibition of PKA should shift phosphorylation states in favor of LTD.
In this study, we demonstrate a marked enhancement of the magnitude of
LTD when PKA is inhibited. This result is in contrast to the
observations of Brandon et al. (1995)
, who reported a
complete block of LTD in control slices by PKA inhibition. The reasons
for these differences are unclear. One explanation could be that
effects of PKA blockade may be age and/or genus dependent. In our
study, we used juvenile rats (14-21 days), whereas older mice (4-6
wk) were used by Brandon et al. (1995)
.
Intracellular infusion of H-89 into postsynaptic pyramidal neurons
produced far less enhancement of LTD than bath application, suggesting
that the effect involves, at least in part, a presynaptic site of
action. However, because the delivery of substances from sharp
microelectrodes is difficult to ensure, we used a 50-fold higher
concentration of H-89 than for bath application and allowed ample
diffusion time (45 min) before stimulation. We previously have found
these methods to be effective in achieving intracellular activity
(Reyes and Stanton 1996
; Stanton and Gage
1996
), but we further verified the presence of inhibition
directly by testing the effect of H-89 infusion on a well-characterized
postsynaptic PKA-dependent response, suppression of the AHP
(Madison and Nicoll 1986
). In control cells,
PKA-dependent inhibition of the AHP by the adenylate cyclase stimulant
forskolin was complete. Forskolin's block was reduced >80%, but not
completely prevented, in cells impaled with electrodes containing 500 µM H-89.
Although postsynaptic infusion of H89 as well as KT5823 support a
presynaptic site for LTD, our PPF data does not. PPF is believed to
reflect enhanced transmitter release due to residual presynaptic
[Ca2+]i, fostering the
idea that manipulations that change release probability should alter
PPF. It is more questionable whether PPF alterations are a sensitive
measure of presynaptic plasticity. Early field potential studies in
field CA1 failed to show changes in PPF after induction of LTP
(Muller and Lynch 1989), whereas some later studies have
reported significant decreases in average PPF ratio
(Kleschevnikov et al. 1997
). It has been suggested that intracellular EPSPs may be more accurate in assessing PPF because they
are less contaminated by polysynaptic events or population action
potentials. Unfortunately, intracellular results have proven just as
equivocal; some showing changes during LTP (Voronin and Kuhnt
1990
) and LTD (Bolshakov and Sieglebaum 1994
),
others do not (Hjelmstad et al. 1997
; Manabe et
al. 1993
). Interestingly, Bertram et al. (1996)
recently
supplied support for an alternative model of PPF that, by depending on
Ca2+ binding domains directly on release apparatus
proteins, could allow for LTP and LTD in the presynaptic terminal that
would not alter PPF. Our data may point to presynaptic targets
insensitive to PPF, or damage the presynaptic hypothesis. Other methods
of evaluating transmitter release will be needed to resolve this question.
The actions of zaprinast appear somewhat different from those of the NO
donor, S-nitroso-N-acetyl penicillamine (SNAP).
We previously have observed that combining submaximal LFS with SNAP elicits a robust, stable LTD (Gage et al. 1997;
Reyes-Harde et al. 1999b
). In the present study,
zaprinast plus submaximal LFS produced only reversible depression,
implying that SNAP, and, hence, NO, possess additional properties that
zaprinast does not. Although nonspecific actions of SNAP are possible,
the concentration we used (100 µM) is below those reported to act
directly on sulfhydryl moieties. An alternative explanation could
involve differences in subcellular localization of PDE, PKA, and PKG.
De Vente et al. (1996)
have shown that both the
magnitude and localization of NO-mediated [cGMP] accumulation in
hippocampus is influenced by isozyme-specific PDE inhibition. Kinase
activity can be restricted spatially, at least for PKA (for review, see
Coghlan et al. 1993
) and also possibly for PKG
(Vo et al. 1998
). Shakur et al. (1993
, 1995
) have shown membrane compartmentalization of a specific
PDE4 isoform, while Whalin et al. (1988a
,b
) have shown
the same for cyclic GMP-stimulated PDE2. In pyramidal cells, it is
possible that inhibiting PDE V might cause local increases in [cGMP]
that activate only a subset of the substrates necessary for LTD. In contrast, NO-activated GC may either activate a different pool of
cyclic GMP or PKG-dependent events and/or stimulate PDE2-mediated hydrolysis of cAMP.
Perhaps of greatest interest, our investigations have culminated
in the discovery of conditions that are both necessary and sufficient
to elicit sustained depression of synaptic efficacy that completely
bypasses the requirement for electrical stimulation, NMDA, or GABA
receptor activation. Interestingly, in the dentate gyrus, Wu et
al. (1998) recently have reported a long-lasting depression of
synaptic strength produced by zaprinast alone, which was, however,
activity-dependent and required metabotropic glutamate receptor (mGluR)
activation. Group II mGluRs are coupled negatively to adenylate
cyclase, providing a potential means of lowering [cAMP]. It is
tempting to speculate that group II mGluRs might effectively serve the
same purpose for LTD in the dentate as H-89 does in our chemical LTD in CA1.
The demonstration that saturating stimulus-induced LTD occludes
chemically induced LTD indicates that these two forms of synaptic depression either act via the same mechanisms or, at some level, converge on shared pathways. Previous studies in our laboratory have
shown that stimulus-induced LTD requires PKG activity, and the
demonstration here that the PKG inhibitor KT5823 blocked chemically induced LTD provides additional support for a convergence. In earlier
studies, we showed that presynaptic ryanodine-sensitive Ca2+ stores (Reyes and Stanton
1996) and activation of presynaptic Ca2+/calmodulin-dependent protein kinase II
(Stanton and Gage 1996
) are both needed to induce LTD.
We recently have found that antagonists of the cGMP-stimulated
messenger cyclic ADP ribose, which releases calcium from stores in sea
urchin eggs (Galione et al. 1991
), also can block the
induction of LTD (Reyes-Harde et al. 1999a
,b
). The role
of this cascade in chemical LTD, and how it modulates glutamate
release, remain to be determined.
In contrast to chemical LTD, the initial reversible depression induced
by zaprinast was not blocked by KT5823. Boulton et al.
(1994) reported this action of zaprinast to be presynaptic, suggesting there must be a cyclic GMP-mediated mechanism for depressing transmitter release that is PKG-independent. Based on the literature, we propose the following. Elevation of [cGMP] activates cyclic GMP-stimulated (type II) PDE, which hydrolyzes cyclic AMP and reduces a
tonic cyclic AMP-mediated enhancement of glutamate release. Studies
supporting this include demonstrations by Doerner and Alger
(1988)
that cyclic GMP can depress hippocampal
Ca2+ currents through a PKG-independent mechanism
and by Broome et al. (1994)
, who found that activation
of presynaptic A1 adenosine receptors, which tonically reduce [cyclic
AMP] and suppress glutamate release, is necessary for zaprinast's
actions. Another possible target is a cyclic nucleotide-gated cation
channel in hippocampus whose tonic activation requires cyclic AMP
(Bradley et al. 1997
). Whether reversible cyclic
GMP-mediated depression is distinct from LTD, or PKG just adds
phosphorylation events that increase the duration of this existing
mechanism, is unknown.
There are multiple mechanisms whereby PKG and PKA might affect
glutamate release (see Fig. 9). Both kinases
can phosphorylate presynaptic channels known to alter transmitter
release. PKA can enhance glutamate release by phosphorylating either
presynaptic Ca2+ channels (Hell et al.
1995) or presynaptic kainate receptors (GluR6)
(Chittajallu et al. 1996
). Conversely, PKG can directly phosphorylate and open K+ channels in hippocampal
neurons (Furukawa et al. 1996
) and indirectly activate
protein phosphatase 2A, which opens K+ channels
in pituitary tumor cells (White et al. 1993
).
Simultaneously reducing Ca2+ conductance through
PKA inhibition and activating K+ channels via PKG
could elicit marked reductions in transmitter release.
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Phosphorylation of synaptic vesicle proteins provide another way
kinases might influence transmitter release. An attractive, but
untested, hypothesis is that PKG may phosphorylate and downregulate a
protein vital to synaptic vesicle mobilization (Sistiaga et al.
1997). Wang and Robinson (1995)
have shown the
existence of >40 PKG substrate proteins that remain uncharacterized
and of unknown function. Several proteins involved in vesicle transport have been identified as potential presynaptic targets for PKA phosphorylation in vitro. These include synapsin1,
-SNAP (
soluble NSF attachment protein), and rabphilin 3A. Injection of
dephosphorylated synapsin1 into squid axons inhibits transmitter
release (Llinas et al. 1985
), but phosphorylated
synapsin has no converse effect, and synapsin1 knockout mice show no
deficits in LTP (Spillane et al. 1995
). Phosphorylation
of rabphilin 3A is required for interaction with another protein,
rab3A, allowing its association with the fusion apparatus and
recruitment of vesicles for exocytosis (Fykse et al.
1995
). Interestingly, in rab3A knockout mice, all electrophysiological parameters are normal in area CA1 except for an
increase in synaptic depression evoked by short stimuli (Geppert
et al. 1994
). In vivo phosphorylation of these proteins has yet
to be shown, leaving the physiological relevance of in vitro studies uncertain.
Recent studies (Kameyama et al. 1998; Lee
et al. 1998
) describe another chemical means for inducing LTD
that used a brief (3 min) bath application of NMDA to hippocampal
slices. It was suggested that this form of LTD is critically dependent
on selective dephosphorylation of postsynaptic GluR1 AMPA receptor
subunits at a PKA-sensitive site and shares mechanisms with
stimulus-induced LTD. It is becoming clear that there are multiple
mechanistically independent forms of LTD (Bolshakov and
Siegelbaum 1994
; Gage et al. 1997
; Oliet
et al. 1997
). Our chemical LTD is activity-independent, bypasses the need for NMDA-receptor activation, and appears to depend
on presynaptic PKG activation and PKA inhibition. Therefore, we
hypothesize that our chemical LTD activates a presynaptic, cyclic-GMP-
and PKG-dependent form of LTD, whereas that described by Lee et
al. (1998)
is a postsynaptic, dephosphorylation-mediated form.
In summary, our data support the hypothesis that in the induction of homosynaptic LTD, cyclic GMP, acting via PKG, is involved in promoting cellular events that are antagonistic to those mediated by activating PKA. The notion that these two cyclic nucleotide second-messenger systems mediate opposing biological signals has been demonstrated in many other tissues. The idea that a specific form of synaptic plasticity depends on the bidirectional control of kinase activity presents an attractive "push-pull" model for the amplification of signal transduction cascades. In this regard, chemically induced LTD should prove useful for the study of the biochemical cascades underlying stimulus-induced LTD.
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ACKNOWLEDGMENTS |
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We thank A. Kyrozis, T. Opitz, A. Peinado, and S. Siegelbaum for helpful discussions.
This work was supported by Whitehall Foundation Grant A98-32 to P. K. Stanton and National Institutes of Health Medical Scientist Training Grant F31GM-16379 to M. Reyes-Harde.
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FOOTNOTES |
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Address for reprint requests: P. K. Stanton, Dept. of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461-1602.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 January 1999; accepted in final form 19 April 1999.
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REFERENCES |
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