Induction of Hippocampal LTD Requires Nitric-Oxide-Stimulated PKG Activity and Ca2+ Release From Cyclic ADP-Ribose-Sensitive Stores

Magali Reyes-Harde,1 Barry V. L. Potter,2 Antony Galione,3 and Patric K. Stanton1

 1Departments of Neuroscience and Neurology, Albert Einstein College of Medicine, Bronx, New York 10461;  2Department of Medicinal Chemistry, School of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY; and  3University Department of Pharmacology, Oxford University, Oxford OX1 3QT, United Kingdom


    ABSTRACT
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ABSTRACT
INTRODUCTION
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REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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), 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).

NO is a rapidly diffusible gas produced by the Ca2+/calmodulin-activated enzyme nitric oxide synthase (Bredt and Snyder 1992). 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.

An important physiological receptor for NO is the heme moiety associated with the family of soluble guanylyl cyclases (NOGCs) (Katsuki et al. 1977; 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.

We also have recently found that intracellular calcium stores play a crucial role in LTD (Reyes and Stanton 1996). 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.

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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 MOmega ) 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.

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). 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.

Drugs were prepared as follows: SNAP (Alexis Corporation) was made into aliquots in powder form and kept frozen (-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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (-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).



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Fig. 2. Blockade of N-methyl-D-aspartate (NMDA) receptors, NO-activated guanylate cyclase (NOGC), cyclic guanosine 3',5' monophosphate-dependent protein kinase (PKG) and Ca2+-dependent Ca2+ release all prevent the ability of SNAP to facilitate LTD. A: time course of experiments (n = 6) where the selective NMDA receptor antagonist D-2-amino-5-phosphonopentanoic acid (D-AP5) (50 µM) was bath-applied for 15 min before 100 µM SNAP plus lfs (1 Hz/400 s; solid bar). AP5 completely prevented SNAP's enhancement of LTD. Each point is the normalized mean ± SE. B: time course of experiments (n = 8) where the selective soluble guanylate cyclase inhibitor 1-H [1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ) (10 µM) was bath applied for 30 min before 100 µM SNAP plus lfs (1 Hz/400 s; solid bar).). ODQ completely prevented SNAP's actions on LTD. Each point is the normalized mean ± SE. C: time course of experiments (n = 6) where the selective PKG inhibitor KT5823 (1 µM) was bath applied for 15 min before 100 µM SNAP plus lfs (1 Hz/400 s; solid bar). KT5823 completely prevented SNAP's actions on LTD. Each point is the normalized mean ± SE D: time course of experiments (n = 8) where ryanodine (10 µM), a selective inhibitor of Ca2+-activated intracellular calcium stores, was bath-applied for 15 min before 100 µM SNAP plus lfs (1 Hz/400 s; solid bar). Ryanodine also prevented SNAP's facilitation of LTD, indicating a corequirement for NO-initiated events and release of Ca2+ from ryanodine-sensitive stores. Each point is the normalized mean ± SE.

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 right-arrow NOGC right-arrow cGMP right-arrow 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.



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Fig. 3. Two different PKG inhibitors block the induction of LTD by a 1 Hz/900 s LFS. A: time course of experiments (n = 8) where the competitive PKG antagonist, Rp-8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate (Rp-8pCPT-cGMP; 10 µm) was bath applied for 30 min before applying a 1 Hz/900 s LFS (solid bar) to one Schaffer collateral input, which completely blocked induction of LTD. Each point is the normalized mean ± SE. B: time course of experiments (n = 5) where a noncompetitive cell-permeant PKG inhibitor, KT5823 (1 µM) was bath applied for 30 min before the induction of LTD by a 1 Hz/900 s LFS (solid bar). Again, LTD was blocked completely by inhibition of PKG. Each point is the normalized mean ± SE.

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).



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Fig. 4. Two selective inhibitors of cADP-ribose block the induction of LTD by LFS. A: time course of LTD of Schaffer collateral-CA1 synaptic transmission in untreated, control hippocampal slices (n = 6) in a continuously recirculating minichamber (~2 ml total volume), induced by a prolonged LFS (2 Hz/10 min; solid bar). Each point for all plots is the normalized mean ± SE. B: time course of experiments (n = 4) where ryanodine (10 µM), a selective inhibitor of Ca2+-activated intracellular calcium stores, was bath applied for 30 min before LFS (2 Hz/10 min; solid bar). Ryanodine prevented the induction of LTD in the recirculating chamber, consistent with our previous findings in a flow-through chamber with 1 Hz/900 s LFS (Reyes and Stanton 1996). C: time course of experiments (n = 4) where 8-Br-cADP-ribose (100 µM), a cell-permeant antagonist of the cADP-ribose-binding site on ryanodine receptors, continuously bathed slices in the recirculating chamber. This concentration of 8-Br-cADP-ribose did not block the induction of LTD by LFS (2 Hz/10 min). D: time course of experiments (n = 4) where a fivefold higher concentration of 8-Br-cADP-ribose (500 µM), was present continuously in the recirculating chamber. This concentration of 8-Br-cADP-ribose was able to partially block the induction of LTD by LFS (2 Hz/10 min). E: time course of experiments (n = 4) where 50 µM 7-deaza-8-Br-cADP-ribose, a more potent, nonhydrolyzable, cell-permeant cADP-ribose antagonist, continously bathed slices in the recirculating chamber. At this concentration, 7-deaza-8-Br-cADP-ribose matched the partial block of induction of LTD by LFS (2 Hz/10 min) exhibited by the low dose of 8-Br-cADP-ribose. F: time course of experiments (n = 4) where doubling the concentration of 7-deaza-8-Br-cADP-ribose (100 µM) completely blocked the induction of LTD by LFS (2 Hz/10 min).

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.



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Fig. 5. Proposed presynaptic, NO-triggered cascade necessary for the induction of LTD. Proposed scheme in which the induction of LTD begins with calcium entry via NMDA receptors and metabotropic glutamate receptor-triggered, inositol triphosphate (IP3)-mediated release of stored calcium, which activates a Ca2+/calmodulin-dependent NO synthase (NOS). NOS produces the retrograde messenger NO, which can diffuse to the presynaptic terminal. NO activates soluble guanylate cyclase (NOGC) to increase [cGMP]. One target of cGMP is PKG, which when activated can phosphorylate many serine/threonine residues. A potential target of PKG is a cyclase (CD38) that produces cADP-ribose from beta -NAD+. Finally, cADP-ribose can modulate release of calcium from intracellular stores by binding to the ryanodine receptors on these stores, making this Ca2+ available to activate CaMKII, presumably leading, by further unknown steps, to reduced transmitter release.

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 beta -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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society