1Departments of Neuroscience and Neurology,
Albert Einstein College of Medicine, Bronx, New York 10461;
2Department of Medicinal Chemistry,
Reyes-Harde, Magali,
Barry V. L. Potter,
Antony Galione, and
Patric K. Stanton.
Induction of Hippocampal LTD Requires Nitric-Oxide-Stimulated PKG
Activity and Ca2+ Release From Cyclic ADP-Ribose-Sensitive
Stores.
J. Neurophysiol. 82: 1569-1576, 1999.
Long-term depression (LTD) of synaptic transmission can
be induced by several mechanisms, one thought to involve
Ca2+-dependent activation of postsynaptic nitric oxide (NO)
synthase and subsequent diffusion of NO to the presynaptic terminal. We used the stable NO donor
S-nitroso-N-acetylpenicillamine (SNAP) to
study the NO-dependent form of LTD at Schaffer collateral-CA1 synapses
in vitro. SNAP (100 µM) enhanced the induction of LTD via a cascade
that was blocked by the N-methyl-D-aspartate
receptor antagonist D-2-amino-5-phosphonopentanoic acid (50 µM), NO guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a]
quinoxalin-1-one (10 µM), and the PKG inhibitor KT5823 (1 µM). We
further show that LTD induced by low-frequency stimulation in the
absence of SNAP also is blocked by KT5823 or
Rp-8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate
(10 µM), cyclic guanosine 3',5' monophosphate-dependent protein
kinase (PKG) inhibitors with different mechanisms of action.
Furthermore SNAP-facilitated LTD was blocked when release from
intracellular calcium stores was inhibited by ryanodine (10 µM).
Finally, two cell-permeant antagonists of the cyclic ADP-ribose binding
site on ryanodine receptors also were able to block the induction of
LTD. These results support a cascade for induction of homosynaptic,
NO-dependent LTD involving activation of guanylyl cyclase, production
of guanosine 3',5' cyclic monophosphate and subsequent PKG activation.
This process has an additional requirement for release of
Ca2+ from ryanodine-sensitive stores, perhaps dependent on
the second-messenger cyclic ADP ribose.
An unresolved question in identifying the
molecular mechanisms underlying long-term depression (LTD) of synaptic
strength, is whether both pre- and postsynaptic changes are involved.
Although it has been demonstrated that the initial events leading to
long-term changes in synaptic strength require a postsynaptic locus of
control mediated by Ca2+ influx (for review see:
Bear and Abraham 1996 NO is a rapidly diffusible gas produced by the
Ca2+/calmodulin-activated enzyme nitric oxide
synthase (Bredt and Snyder 1992 An important physiological receptor for NO is the heme moiety
associated with the family of soluble guanylyl cyclases (NOGCs) (Katsuki et al. 1977 We also have recently found that intracellular calcium stores play a
crucial role in LTD (Reyes and Stanton 1996 Data from this paper are from a thesis submitted in partial fulfillment
of the requirements for the degree of Doctor of Philosophy in the Sue
Golding Graduate Division of Medical Sciences, Albert Einstein College
of Medicine.
Experiments were performed on transverse hippocampal slices
prepared from 14- to 21-day-old Sprague-Dawley rats of either sex
decapitated under deep ether anesthesia. The hippocampus plus entorhinal cortex was dissected out, and slices 400-µm thick were cut
simultaneously using a spring-loaded mechanism ("egg slicer") that
rapidly forces a parallel grid of 20-µm-diam wires through the
tissue. Slices recovered for 1-2 h in a humidified, oxygenated (95%
O2-5% CO2) Haas-style
interface recording chamber perfused at 3 ml/min with artificial
cerebrospinal fluid (ACSF) at 33°C. ACSF composition (in mM) was 126 NaCl, 26 NaHCO3, 1.25 NaH2PO4, 5 KCl, 2 CaCl2, 2 MgCl2, and 10 D-glucose, pH 7.4. For experiments using cyclic ADP-ribose
antagonists, we used a special minichamber that recirculated a 2 ml
volume of ACSF at the same temperature (33°C).
Two separate Schaffer collateral-commissural axon populations were
isolated by placement of stimulating electrodes in stratum radiatum on
opposite sides of the recording site, verified as separate inputs by a
lack of paired-pulse facilitation between them (50-ms interstimulus
interval), and alternately stimulated each 30 s throughout the
experiment using bipolar stainless steel electrodes (Frederick-Haer;
150-µs DC square pulses). Extracellular recording electrodes
(RE = 2-5 M LTD was evoked by a single low-frequency stimulus train of 900 pulses
(150 µs) delivered at a frequency of 1 Hz (LFS) (Dudek and
Bear 1992 Drugs were prepared as follows: SNAP (Alexis Corporation) was
made into aliquots in powder form and kept frozen ( NO donor SNAP enhances homosynaptic LTD
Figure 1A illustrates the
response of control, untreated hippocampal slices to LFS. Prolonged LFS
(1 Hz/900 s) of Schaffer collateral axons produced a robust, stable LTD
of synaptic strength in field CA1 (
ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
), presynaptic changes in
neurotransmitter release properties also have been associated with LTD
(Bolshakov and Siegelbaum,1994
; Oliet et al.
1996
). These constraints require a retrograde messenger to
provide presynaptic terminals with information about postsynaptic
activity. Of several candidates, arachidonic acid and nitric oxide (NO)
are two membrane-permeant substances that have been suggested as
potential retrograde messengers. Arachidonic acid, generated from the
degradation of membrane phospholipids by phospholipase A2, has been
suggested to play such a role in LTD (Bolshakov and Siegelbaum
1995
). However, a selective inhibitor of
PLA2 has been shown to be without effect on LTD
(Stanton 1995
).
). The hypothesis that
this unstable, free-radical species functions as an intercellular
messenger that generates guanosine 3',5' monophosphate (cGMP) in
response to stimulation of neuronal glutamate receptors was first
proposed by Garthwaite and Garthwaite (1987)
.
Izumi and Zorumski (1993)
first supplied evidence of a
role for NO in the induction of LTD at Schaffer collateral-CA1 synapses
in the hippocampus. We (Gage et al. 1997
) pursued this
line of investigation and found that pairing the NO donor,
S-nitroso-N-acetyl penicillamine (SNAP), with a
weak low-frequency (1 Hz/400 s) stimulus, promotes LTD in rat
hippocampal slices.
; Southam and Garthwaite
1993
). Our laboratory recently has demonstrated that NOGC can
play a necessary role in the induction of LTD in the hippocampus
(Gage et al. 1997
). In these studies, we showed that the
specific NOGC inhibitor 1H-[1,2,4] oxadiazolo [4,3-a]
quinoxalin-1-one (ODQ), developed by Garthwaite et al.
(1995)
, blocks the induction of LTD produced by prolonged low-frequency stimulation (1 Hz/15 min), but that postsynaptic injection of inhibitor did not produce blockade. These findings suggest
that an increase in presynaptic [cGMP] is necessary for the induction
of LTD. Cyclic GMP has many potential direct targets, including
phosphodiesterases (PDEs), kinases (PKG), and cyclic-nucleotide gated
channels (CNGCs) (for review, see MacFarland, 1995
). In the present work, we further investigate pathways leading to
NO-dependent LTD, focusing particularly on the role of PKG.
). One of these stores is sensitive to the plant alkyloid ryanodine, and our
studies suggested that this calcium store has a presynaptic locus of
action, consistent with immunohistochemical evidence from Sharp
et al. (1993)
. Here we further test whether NO-triggered LTD
also depends on intracellular ryanodine-sensitive calcium stores.
Ryanodine receptors are regulated by a complex array of endogenous
messengers including Ca2+, calmodulin, cyclic ADP
ribose, palmitoyl-CoA, Mg2+, spermine, and other
polyamines (Dousa et al. 1996
). Of these messengers,
cyclic ADP-ribose is known to promote release from ryanodine-sensitive
calcium stores in sea urchin eggs through NO-cyclic GMP-dependent
cascades (Willmott et al. 1996
). We therefore investigated whether regulation of ryanodine receptors by the second-messenger cyclic ADP-ribose (cADPR) might play a role in the
induction of LTD.
METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
) were filled with 2 M
NaCl. We adjusted stimulus intensity so that the amplitude of each
population EPSP was 50% of maximum (>2 mV) as determined by an
input/output curve.
). A "submaximal" lfs consisted of 400 pulses at 1 Hz. For experiments performed in the recirculating minichamber, the
maximal LFS was delivered at a frequency of 2 Hz for 10 min (1,200 pulses). As a measure of excitatory monosynaptic strength, the maximum
initial negative slopes of the field excitatory postsynaptic potentials
(EPSPs) were calculated using a 6-point interpolation least-squares
linear regression method (DataWave Technologies software). Each point
plotted is from slopes normalized to pre-LFS baselines and averaged
over all experiments, ±SE. The change in synaptic strength was
measured as percent change between average baseline values over the 10 min immediately preceding LFS, and the average of 10 points spanning
the 30-min post-LFS time point. Statistical significance was
established using a two-tailed Student's t-test.
20°C) until time
of use. Just before bath application, it was diluted in warm distilled
H2O, sonicated, and then diluted to a final concentration of 100 µM in ACSF. ODQ (Biomol), KT5823 (Biomol), Ryanodine (RBI) were dissolved in DMSO as 1,000× stocks. AP5 (Tocris Cookson), Rp-8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMP; Biolog), 8-Bromo-cADPR (Sigma) and
7-deaza-8-Bromo-cADPR (synthesized by B.V.L. Potter and A. Galione)
were dissolved in distilled H2O as 1,000× stocks. All
drugs were made into aliquots and kept frozen (
20°C), until time of
use unless otherwise noted.
RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
35.3 ± 4.0%;
n = 16; P < 0.05, paired
t-test, 30-min post-LFS compared with pre-LFS baseline EPSP
slopes). In contrast, a shorter stimulus duration of 400 s, termed
submaximal lfs, elicited no statistically significant depression from
baseline, as shown in Fig. 1B (
9.5 ± 5.4%;
n = 9; P > 0.05, paired
t-test, compared with pre-lfs baseline EPSP slopes).
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Fig. 1.
Stable nitric oxide (NO) donor,
S-nitroso-N-acetylpenicillamine (SNAP),
enhances the ability of a submaximal low-frequency stimulus (lfs) to
induce homosynaptic long-term depression (LTD). A: time
course of maximal LTD of Schaffer collateral-CA1 synaptic transmission
in untreated, control hippocampal slices induced by a prolonged LFS (1 Hz/900 s; solid bar). Each point is the normalized mean ± SE of
responses from 16 slices. B: in contrast, stimulation of
Schaffer collaterals with a submaximal low frequency stimulus (lfs; 1 Hz/400 s; solid bar) did not evoke full LTD. Each point is the
normalized mean ± SE of 9 slices. C: time course
of experiments (n = 8) where the stable NO-donor,
SNAP (100 µM), was bath applied for 20 min before lfs (1 Hz/400 s;
solid bar). Significantly greater LTD was evoked in the presence of
SNAP. Each point is the normalized mean ± SE. D:
time course of SNAP-enhanced LTD at 1 of 2 independent Schaffer
collateral inputs (S1 and S2; see inset) alternately
stimulated every 60 s. After bath-application of SNAP, a 1 Hz/400
s lfs was applied only to S1 (closed circles), while S2 (open circles)
served as control. SNAP's actions in promoting LTD were homosynaptic.
Each point is the normalized mean ± SE of 4 slices.
To directly test the effects of activating NO-dependent cascades
on the response to submaximal lfs, we used the stable NO-donor compound
SNAP. Although high concentrations (1 mM) of SNAP have been reported to
produce a reversible depression of Schaffer collateral-CA1 EPSPs, lower
concentrations (100 µM) do not directly affect EPSP slopes
(Boulton et al. 1994) (Fig. 1D). However,
pairing a low concentration of SNAP (100 µM) with a submaximal lfs (1 Hz/400 s) elicited LTD, as shown in Fig. 1C (
28.7 ± 6.9%; n = 8), which is statistically significant when
compared with 1 Hz/400 s stimulation alone (P < 0.05, Student's t-test). Because the entire slice was bathed in
SNAP, we ensured that the LTD that was induced in SNAP was homosynaptic
by monitoring a control, unstimulated input in the same slice. Figure
1D illustrates the time course of experiments (n = 4) in which two distinct synaptic inputs, S1 and
S2 (see inset schematic), were stimulated alternately. Only
the input pathway (S1), which received submaximal lfs (1 Hz/400 s),
showed significant LTD (
28 ± 3.6%), whereas the unstimulated
path (S2) remained stable at baseline values (
4.4 ± 2.0%).
SNAP-facilitated LTD requires NMDA receptor activation
Because NO can be produced secondary to the activation of
NMDA glutamate receptors that gate influx of Ca2+
into the postsynaptic neuron (Garthwaite et al. 1989)
and because blockade of NMDA receptors can block the induction of pure
stimulus-evoked LTD (Dudek and Bear 1992
; Wexler
and Stanton 1993
), we tested whether there is still a
requirement for activation of NMDA receptors when NO is supplied
exogenously. In the experiments shown in Fig. 2A, we bath applied the
selective NMDA receptor antagonist
D
2-amino-5-phosphonopentanoic acid (D-AP5; 50 µM; open bar) 15 min before the coapplication of SNAP (100 µM;
filled bar) plus submaximal lfs (1 Hz/400 s; solid bar). Blockade of
NMDA receptors completely reversed the facilitatory actions of SNAP
(+3.9 ± 12.6%; n = 6; P < 0.05, Student's t-test compared with submaximal lfs + SNAP).
|
SNAP enhances LTD through activation of NOGC
Next, we focused on identifying the downstream messengers involved
in the NO-initiated presynaptic cascade underlying this form of LTD,
which we will refer to as SNAP-LTD. We previously have shown that
presynaptic NOGC is necessary for the induction of LTD by a prolonged
LFS (1 Hz/900 s) at Schaffer collateral-CA1 synapses (Gage et
al. 1997). SNAP has been shown to elevate [cGMP] dose-dependently in the hippocampus in a manner that is sensitive to
inhibition by hemoglobin (Boulton et al. 1994
). To
assess whether SNAP-LTD also requires activation of soluble guanylyl
cyclases, we used the new selective NOGC inhibitor 1H-[1,2,4]
oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ; 10 µM), which has an
IC50 of ~20 nM (Garthwaite et al.
1995
). As shown in Fig. 2B, bath application of ODQ
(10 µM; open bar) before SNAP (100 µM; filled bar) completely
abolished the facilitatory effects of SNAP (
0.9 ± 6.1%;
n = 8; P < 0.05, Student's
t-test, compared with SNAP + submaximal lfs), consistent
with the conclusion that SNAP-initiated cascades necessary for LTD are
dependent on the activation of NOGC rather than on another, nonspecific
action of SNAP.
PKG is a downstream effector of NO-dependent LTD
We (Gage et al. 1997) and others (Zhuo et
al. 1994
) have shown that bath application of
membrane-permeable cGMP analogues also can facilitate the induction of
LTD when paired with a submaximal LFS. Cyclic GMP could be acting
directly on cyclic nucleotide-gated ion channels to activate or inhibit
phosphodiesterases or to activate cyclic GMP-dependent protein kinases
(PKG). To test the necessity for PKG activation for SNAP-LTD, we used
the selective PKG inhibitor KT5823 (1 µM), which competes for the
ATP-binding site on PKG (Kase et al. 1987
). As shown in
Fig. 2C, bath application of KT5823 before SNAP plus
submaximal lfs (1 Hz/400 s) completely blocked SNAP-LTD (4.5 ± 4.2%; n = 6). EPSP slopes 30 min post lfs were not
significantly different from those evoked by lfs alone
(P > 0.05, unpaired t-test compared with
submax lfs). Thus PKG does appear to be one necessary downstream
effector in the cascade initiated by NO activation of NOGC and
production of cyclic GMP.
Ca2+ release from ryanodine-sensitive stores is a corequirement for SNAP-LTD
There are innumerable potential targets for PKG phosphorylation of
serine/threonine residues. Previous work by our laboratory suggests
that a presynaptic ryanodine-sensitive Ca2+ store
is necessary for the induction of LTD by full LFS (Reyes and
Stanton 1996). Such a calcium source also could be a potential target of the NO-initiated cascade and therefore might be downstream of
both NOGC and PKG. As a test of this hypothesis, we bath-applied the
selective inhibitor ryanodine (10 µM; open bar), which blocks ryanodine-receptor channels and prevents Ca2+
release from this pool, before applying SNAP (100 µM; filled bar)
plus lfs. Figure 2D illustrates these experiments, in which ryanodine completely blocked the induction of SNAP-LTD (
11 ± 10.9%; n = 8; P < 0.05, Student's
t-test, compared with SNAP + submaximal lfs). This supports
our hypothesis that a presynaptic LTD cascade mediated by NO
NOGC
cGMP
PKG also requires release of Ca2+
from Ca2+ -mediated ryanodine-sensitive stores.
PKG is also necessary for the induction of pure stimulus-evoked LTD
Although SNAP might be expected to act via a NO-cyclic GMP-PKG
mechanism, the same might or might not be true for pure stimulus-evoked LTD. Therefore we also assessed the necessity for PKG activity for the
induction of LTD by full LFS (1 Hz/900 s) by using two mechanistically
distinct cell-permeant inhibitors of PKG. In Fig. 3A, we bath applied
Rp-8pCPT-cGMP (10 µM; open bar), a cyclic GMP analogue that derives
its inhibitory actions from the replacement of equatorial cyclic
phosphate oxygens with sulfur, making it a competitive inhibitor that
is resistant to phosphodiesterase-mediated hydrolysis and is highly
lipophilic (Ki = 0.5 µM)
(Butt et al.,1994). Similar to SNAP-LTD, inhibition of
PKG with Rp-8pCPT-cGMP blocked the induction of LTD (
4.5 ± 5.5%; n = 8) by a 1 Hz/900 s LFS (solid bar) as
compared with control LTD (P < 0.05, Student's t-test). In addition, we also tested the PKG inhibitor
KT5823, which blocks PKG through a mechanism distinct from
Rp-8-pCPT-cGMP. As shown in Fig. 3B, bath application of 1 µM KT5823 (open bar) 30 min before LFS (solid bar) also blocked the
induction of LTD by full LFS (4.2 ± 6.1%; n = 5;
P < 0.05, Student's t-test compared with
control LTD). Taken together, our data do support a requirement for PKG
activity in both LTD induced by a prolonged LFS and SNAP-LTD.
|
Cyclic ADP-ribose may be an endogenous activator of ryanodine receptors necessary for LTD
Cyclic ADP-ribose, discovered by Lee and associates
(1989), is a cyclic adenine nucleotide that can release
Ca2+ from stores in a variety of cells
(Currie et al. 1992
; Koshiyama et al.
1991
; Takasawa et al. 1993
), suggesting it may
act as an endogenous second messenger that mobilizes calcium from
ryanodine-sensitive intracellular stores in neurons. We tested the
hypothesis that this messenger might play a role in the induction of
LTD by using two distinct cell-permeant cyclic ADP-ribose analogues
that competitively antagonize its actions on calcium release
(Sethi et al. 1997
).
Because of the fact that a very limited supply of each antagonist was
available to us, we employed an interface recording chamber which
continuously recirculated a small volume (2 ml) of ACSF. Figure
4 illustrates experiments in this
chamber. In Fig. 4A, we demonstrate that LTD could be
induced that was indistinguishable from previous LTD (40.0 ± 5.9%, n = 8; P > 0.05 unpaired
Student's t-test) although the optimal stimulation
frequency was 2 Hz rather that 1 Hz (solid bar; 2 Hz/10 min). Figure
4B demonstrates that the bath application of 10 µM
ryanodine (open bar) was still able to block LFS-induced (solid bar)
LTD in the recirculating chamber (
14.9 ± 12%,
n = 4; P < 0.05, Student's
t-test compared with control LFS), consistent with previous
results (Reyes and Stanton 1996
).
|
Figure 4, C-F, illustrates the effect on LTD of the
two cell-permeant cyclic ADP-ribose antagonists, 8-Bromo-cyclic
ADP-ribose and 7-deaza-8-Bromo-cyclic ADP-ribose. In Fig.
4C, 100 µM 8-Br-cADPR was present in the bath throughout
the experiment (open bar). This concentration of drug was unable to
block the induction of LTD (36.1 ± 4.4%, n = 4; P > 0.05, Student's t-test compared with control LTD). However, a fivefold higher concentration (500 µM;
Fig. 4D) did partially block the induction of LTD
(
24.5 ± 4.8%, n = 4; P < 0.05, Student's t-test).
Using the newly developed, more potent analogue, 7-Deaza-8-Br-cADPR,
which is nonhydrolyzable as well as lipophilic (Sethi et al.
1997), a low concentration (50 µM; Fig. 4E)
produced a partial block of LTD (
14.7 ± 6%, n = 4; P < 0.05, Student's t-test compared
with control LTD). Furthermore Fig. 4F illustrates the effects of 100 µM 7-deaza-8-Br-cADPR, which completely blocked the
induction of LTD, uncovering a modest potentiation after LFS (+9.1 ± 4.5%; n = 4; P < 0.05 Student's
t-test). Taken together, these experiments support a crucial
role for a cADP ribose-binding site (perhaps on ryanodine receptors) in
the induction of LTD.
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DISCUSSION |
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LTD is a long-lasting decrease in the strength of synaptic
transmission. Although the locus of initial events, such as
Ca2+ influx, leading to the induction of LTD is
usually postsynaptic (Mulkey and Malenka 1992),
presynaptic changes in neurotransmitter release also have been
associated with LTD (Bolshakov and Siegelbaum 1994
;
Oliet et al. 1996
) This leads to the hypothesis that a
retrograde messenger may be required to "inform" the presynaptic
terminal that the requirements for LTD have been met postsynaptically. One putative retrograde messenger is NO. Here we supply evidence for a
presynaptic cascade (Fig. 5), initiated
by NOGC, production of cGMP, and activation of PKG, which is critical
to the induction of LTD.
|
NO is a membrane permeable gaseous messenger that can act on any presynaptic terminal within the three-dimensional volume of its diffusional space, limited by chemical breakdown. If NO is a retrograde messenger mediating LTD, it would have to evoke a change in only those terminals that had been previously active when the postsynaptic conditions for LTD induction were met. In this study, we found that the stable NO donor, SNAP, evoked LTD only when paired with a weak lfs (1 Hz/400 s), suggesting that additional stimulus-induced events of a homosynaptic nature were involved. Even though NO was provided exogenously, when it was combined with a weak lfs, it produced LTD only at those synapses that had been stimulated.
The excitatory neurotransmitter glutamate, acting at NMDA receptors,
can stimulate the formation of NO by triggering an influx of
Ca2+ that binds to calmodulin to activate NO
synthase (NOS) (Bredt and Snyder 1989). Because in our
SNAP experiments we provided NO to the presynaptic terminal
exogenously, we tested whether NMDA receptor-activated cascades were
still required for LTD. Our observations that the NMDA antagonist, AP5,
blocked SNAP-LTD imply that there are still
Ca2+-triggered cascades, in addition to NO,
required. Our results with the NO-donor, SNAP, are consistent with
those of Zhuo et al. (1994)
, who found that pairing a
0.25-Hz LFS with NO-saturated ACSF elicited LTD in guinea pig
hippocampal slices. However, in contrast with our findings, they
reported no effect of AP5 on their form of LTD. The differences in
these results may be a function of differing experimental conditions.
Our finding that exogenous SNAP did not preclude the requirement for
NMDA receptor activation suggests that a postsynaptic component
activated by the lfs still is required, in concert with a presynaptic
NO-initiated cascade, for LTD. Because all our other studies (see
Gage et al. 1997
; Reyes and Stanton 1996
)
favor a presynaptic locus for the NO-triggered cascade, NMDA receptor
activation may be necessary for a postsynaptic component of LTD.
However, recent evidence suggests that there also may be presynaptic
NMDA receptors capable of interacting with NO-activated cascades
(Johnson et al. 1996
).
A principal physiological target of NO is soluble guanylate cyclase
(Southam and Garthwaite 1993). Until recently, there
have been no potent and selective inhibitors of this enzyme. Two
compounds often used as guanylyl cyclase inhibitors are LY-83583 and
methylene blue. However, LY-83583 and methylene blue are both more
potent NOS inhibitors, and methylene blue also generates superoxide
anions, making studies with these compounds ambiguous at best
(Luo et al. 1995
). Fortunately, the more-selective new
NOGC inhibitor ODQ does not affect NOS activity or synaptic glutamate
receptor function, as assessed in hippocampal slices, nor does it
chemically inactivate NO (Garthwaite et al. 1995
).
Using ODQ to selectively inhibit NOGC, we have demonstrated
(Gage et al.,1997) that LTD induced by a prolonged LFS
(1 Hz/900 s) depends on activation of NOGC. In these studies, infusion
of ODQ into postsynaptic CA1 pyramidal neurons did not block LTD, indicating a presynaptic locus of NOGC. Here, we provide evidence that
SNAP-enhanced LTD also depends on the activity of NOGC. Thus NO's
primary target in LTD is most likely presynaptic soluble guanylate
cyclase, which when activated produces the second-messenger cyclic GMP.
Immunohistochemical evidence also supports the idea that NOS and NOGC
are transsynaptic functional partners. These two enzymes have
complementary, rather than identical distributions, NOS often is found
in postsynaptic structures, whereas cyclic GMP accumulates in
presynaptic elements and fibers (Southam and Garthwaite
1993
).
While cGMP has many potential targets, we demonstrate here that one necessary target is likely to be cGMP-dependent protein kinase (PKG). Blockade of PKG activity by either of two inhibitors (Rp-8-pCPT-cGMP and KT5823) with differing mechanisms of action, reversed both SNAP-LTD, and LTD induced by a full 900-s LFS.
In previous work, we have demonstrated an involvement of anatomically
distinct pools of intracellular stored Ca2+ in
the induction of LTD (Reyes and Stanton 1996). On the
presynaptic side, endosomal Ca2+ stores gated by
ryanodine receptors (RyR) appear to play an important role. One
downstream target of PKG phosphorylation in the presynaptic terminal
could be this store. Using the specific antagonist ryanodine to block
release from this pool, we found that SNAP-LTD was blocked. From this,
we conclude that either intracellular calcium stores, sensitive to the
plant alkyloid ryanodine, are themselves a target of a presynaptic
NO-initiated cascade, or release from these stores is required in
conjunction with activation of this cascade.
In the former case, PKG could act by directly phosphorylating RyRs to
modify their sensitivity to a Ca2+ trigger
(Hain et al. 1995). Alternatively, PKG also might
phosphorylate another target that has the ability to modify the
sensitivity of RyRs. One potential target is CD38 (Galione et
al. 1993
), a recently identified hydrolase/cyclase which
converts
-NAD+ to cyclic ADP-ribose. Cyclic
ADP-ribose is a newly identified second-messenger that has been shown
to enhance release of Ca2+ from
ryanodine-sensitive stores in sea urchin eggs (Galione et al.
1991
) via NO, PKG, and cGMP (Willmott et al.
1996
). It is not yet known whether cGMP, via PKG, acts to
facilitate the induction of LTD by generation of cyclic ADP-ribose in
presynaptic terminals. For that matter, it is still an open question
whether cyclic ADP-ribose acts directly on RyRs. Ryanodine has been
shown to elicit release of calcium even when cyclic ADP-ribose-induced
release was inhibited effectively by 8-amino-cADP-ribose
(Walseth and Lee 1993
). Whether by a direct or indirect
action on RyRs, our data suggest that inhibition of a cyclic ADP-ribose
binding site does block the induction of LTD.
In the case where release from intracellular calcium stores is required
in conjunction with NO, we have opportunities for coincidence detection
on both presynaptic and postsynaptic sides of the synapse. Perhaps the
role of LFS is to activate release of calcium from presynaptic stores
and, only when this calcium coincides with activation of the
NO-triggered cascade, is the criteria for a presynaptic modification
leading to LTD fulfilled. In this regard, it is interesting to note
that Ca2+/calmodulin exerts a biphasic regulation
of RyRs, where high concentrations of Ca2+
inhibit and lower levels activate these receptors (Lee et al. 1994), and that NOGC also is subject to negative feedback
regulation by Ca2+ (Garbers and Lowe
1994
). These observations are consistent with the idea that a
window of presynaptic calcium concentration is needed to trigger LTD.
One potential target of the calcium released from presynaptic stores
may be Ca2+/calmodulin-dependent protein kinase
II, which we have shown plays a role in the induction of LTD
(Stanton and Gage 1996
). At the same time, it also has
been shown that postsynaptic calcium stores activated by metabotropic
glutamate receptors are localized in dendritic spines (Takechi
et al. 1998
) and that inhibition of postsynaptic phospholipase
C can block the induction of LTD (Reyes-Harde and Stanton
1998
).
The majority of phosphoproteins known to be relatively specific
substrates for PKG are associated with either phospholipids or the
cytoskeleton (Wang and Robinson 1995). Interestingly,
Sistiaga et al. (1997)
have found that altering
intrasynaptosomal cGMP results in an inhibition of glutamate
exocytosis. This depression was neither related to a decrease in
[ATP], nor to a reduction in the entry of Ca2+
into nerve terminals, but consistent with a PKG-dependent inhibition of
a delayed component of release likely to account for vesicle mobilization. Taken together with our current findings, the next step
will be to identify those substrates of PKG that could play a role in
synaptic vesicle release and LTD. One of these targets may be the
cyclase, which produces the second-messenger cyclic ADP-ribose, but
others may well be proteins that are direct members of the
vesicular-release apparatus.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge the helpful discussions of T. Opitz and L. Santschi.
This research was supported in part by Whitehall Foundation Grant A98-32 to P. K. Stanton and National Institute of General Medical Sciences Medical Scientist Training Grant F31GM-16379 to M. Reyes-Harde.
Correspondence should be addressed to P. K. Stanton, Albert Einstein College of Medicine, Dept. of Neuroscience, Kennedy Center Room B33, 1300 Morris Park Ave., Bronx, NY 10461.
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FOOTNOTES |
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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 12 February 1999; accepted in final form 31 March 1999.
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REFERENCES |
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