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Department of Physiology and Program in Neuroscience, University of Maryland School of Medicine, Baltimore, Maryland 21201
Submitted 28 July 2003; accepted in final form 2 October 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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80% of control levels, after washout lasting as long as 2 h. The lasting suppression had several properties in common with low-frequency stimulation induced long-term depression (LFS-LTD), including an ability to depotentiate long-term potentiated responses. However, DHO-LTD was insensitive to blockade of N-methyl-D-aspartate or mGlu receptors or to inhibitors of protein kinase C or p38 MAP kinase. DHO-LTD did not co-occlude with LFS-LTD and therefore appears to represent a novel form of LTD. Interestingly, DHO-LTD could be prevented by pretreating slices with iberiotoxin, the selective blocker of large, Ca2+-dependent K+ channels ("big K," BK channels), although this toxin did not affect basal fEPSPs. Certain pathological conditions, including hypoxia and ischemia, are associated with a decrease in Na,K-pump activity and hence DHO-LTD may serve as a model for the effects on neuronal function in these conditions. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Linden 1994). LTD can be induced by a number of induction protocols, including synaptic and pharmacological stimulation, many of which are dependent on the age of the animal (Kemp et al. 2000). Synaptic LTD is induced by delivery of a low-frequency tetanic stimulation (LFS). Chemical LTD can be induced by brief application of agonists of N-methyl-D-aspartate (NMDA) (Kamal et al. 1999; Lee et al. 1998) or metabotropic glutamate (mGlu) receptors (Camodeca et al. 1999; Fitzjohn et al. 1999) in the absence of synaptic stimulation. The mGluR-dependent form of LTD can also be induced in CA1 of hippocampus by LFS (Oliet et al. 1997) or a paired-pulse stimulus train (Huber et al. 2000; Kemp et al. 2000). There is a similar diversity in LTD expression mechanisms, which can involve protein synthesis (Zhu et al. 2002), protein phosphatase activation (Mulkey et al. 1993), or internalization of glutamate receptors (Beattie et al. 2000; Morishita et al. 2001; Snyder et al. 2001). LTD involves prolonged stimulation in vitro, and yet it is not clear under what circumstances this would occur in vivo. Prolonged release of agonist and activation of intracellular second-messenger systems does occur under pathophysiological conditions, so the possibility of LTD induction under pathophysiological conditions is an important issue.
One consequence of brain injuries that cause hypoxia or anoxia is a reduction in the activity of Na,K-ATPases (the Na,K-pump) (Haddad and Jiang 1993; Lees 1991; McNamara 1994). Inasmuch as such injuries can cause memory loss and other cognitive disturbances, it is of interest to inquire into the synaptic mechanisms that might be responsible. Ross and Soltesz found that early traumatic injury (2000) or high-frequency stimulation (2001) altered the excitability of dentate gyrus interneurons by causing a reduction in Na,K-pump activity. Partial inhibition of Na,K-pump activity by the low-affinity ouabain analogue, dihydroouabain (DHO) increases neuronal excitability (McCarren and Alger 1987) but in addition suppresses fEPSPs and EPSPs (Vaillend et al. 2002). Importantly, we found that the low concentrations of DHO used did not significantly affect intrinsic cell properties or extracellular [K+] because they only affect the ouabain-sensitive, 
2 and 3, isoforms of the Na-pump (Berrebi-Bertrand et al. 1990; Sweadner 1989) that are not primarily responsible for the "housekeeping" functions of the cells. Hence the suppression of fEPSPs is likely to reflect factors specific to the process of synaptic transmission. The increased excitability represented an increase in EPSP-spike coupling, but we did not investigate the fEPSP depression that began when DHO was present in the bath. The acute fEPSP suppression was accompanied by suppression of fiber volleys, but the lasting suppression was not explored. Although it resembled LTD, no comparison of the two phenomena was made. The purpose of the present work was to determine if the lasting, DHO-induced suppression of excitatory synaptic potentials represents a form of LTD and to begin to investigate the cellular mechanisms involved.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Male Sprague-Dawley rats aged 30–60 days (Charles River Laboratories) were deeply sedated with halothane and decapitated in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine. The brain was removed, and hippocampi were dissected out and cut into 400-µm-thick transverse hippocampal slices on a Vibratome (Series 1000; Technical Products International) as previously described (McCarren and Alger 1987). After recovery for 1 h at room temperature, slices were transferred to a submersion chamber and perfused with saline (29–31°C) at a flow rate of 0.5–1 ml/min (Nicoll and Alger 1981). The saline comprised (in mM): 120 NaCl, 3 KCl, 2 MgSO4, 1 NaH2PO4, 25 NaHCO3, 2,5 CaCl2, and 10 glucose and was saturated with 95% O2-5% CO2 (pH 7.4).
Field potentials were recorded from stratum radiatum and/or s. pyramidale of CA1 with glass microelectrodes broken to a tip diameter of 4–7 µm and filled with 1 M NaCl or with saline (5–15 M
). The stimuli were delivered by bipolar stimulating electrodes in s. radiatum at 0.05 Hz and consisted of square pulses 50- to 100-µs duration. Fiber volleys and antidromic population spikes were evoked by s. radiatum and alvear stimulation, respectively. Signals were digitized at 10 kHz with an A/D interface (Digidata 1200, Axon Instruments) and analyzed with pCLAMP (Axon Instruments).
Data analysis
The initial slope of the fEPSP (mV/ms) was measured within the first millisecond of the response immediately after the negative peak of the fiber volley. Fiber volley amplitude (mV) was determined from the difference between the initial positive and negative peaks. In the isolated fiber volley experiments, TTX (1 µM) was applied at the end of the experiment for subtraction of the stimulus artifact. Antidromic population spike amplitude (mV) was determined by taking the difference between baseline potential before the stimulus artifact and the negative peak of the response.
To quantify the results, values from 15 consecutive responses in the experimental condition were divided by the mean of 15 consecutive responses in the baseline condition. The magnitude of the change in the response was expressed as the mean percent of control before DHO, high- or low-frequency stimulation (HFS or LFS; means ± SE). Long-term potentiation (LTP) was quantified 35–40 min after HFS; as noted LTD was quantified from 25 to 120 min after DHO application or after LFS. To compare the magnitude of the suppressive effect of DHO in naïve and potentiated slices directly, responses in the depotentiation experiments were also normalized to the mean response 40 min after HFS. Comparisons of mean values were obtained with Student's paired or unpaired t-test or one- and two-way repeatedmeasures ANOVAs.
Materials
Stock solutions of DHO (10 mM; Sigma) were prepared in deionized water and bath applied at a final concentration of 20 µM for 15 min in all experiments except where otherwise noted. All other drugs were prepared as concentrated stock solutions and diluted by 1:1,000 in saline. Water-based stock solutions included 2-amino-5-phosphonovaleric acid (APV, 50–100 µM; Sigma), LY341495 (100 µM; Tocris), 1,2,3,4-tetrahyrdo-6-nitro,2,3, dioxobenzo(f)quinoxaline-7-sulfonamide (NBQX, 10 µM; Sigma), tetrodotoxin (TTX, 1 µM; Sigma), iberiotoxon (IbTX, 100 nM; Tocris), and tetraethylammonium (TEA, 2 mM; Sigma). DMSO-based stock solutions included AM-251 (4 µM; Tocris), SB 203580 (25 µM; Tocris), and GF 109 20X (bisindoylmaleinmide 1; BIS-1, 5 µM; Tocris). The final concentration of DMSO was 0.01% (vol/vol).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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reported that a 15-min bath application of DHO suppressed excitatory synaptic responses that did not fully recover after DHO had washed from the slice for 
20 min. This effect could not obviously be explained by an incomplete removal of DHO because the DHO effects on the Na,K-pump reversed with a time constant of 12 min (cf. Fig. 1B of Vaillend et al. 2002). We now report that fEPSPs do not recover even if the wash from DHO is prolonged to 2 h. For all 17 slices, fEPSP slope was only 81.7 ± 2.1% of control amplitude after 50 min of wash (P < 0.001) and, in 4 slices that were followed longer, fEPSP initial slope was 80.2 ± 2.4% of control after 120 min of wash (Fig. 1A, 2 and 3; P < 0.001). Hence the long-term suppression induced by DHO does not depend on the continued presence of DHO in the tissue, and we refer to it as DHO-LTD. Conventional LTD (LFS-LTD) induced by a low-frequency stimulation of the Schaffer collaterals (1 Hz for 900 pulses) (Dudek and Bear 1992; Mulkey and Malenka 1992) suppressed the fEPSP initial slope to 80.3 ± 2.6% of baseline 50 min after the train (n = 5, P < 0.05, data not shown). Thus the magnitude of DHO-LTD was not significantly different from that of LFS-LTD (DHO-LTD: n = 13 vs. LFS-LTD: n = 5, P > 0.05) and was similar to it in duration.
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To determine if DHO-LTD shared similarities with conventional LTD, we asked if DHO-LTD could depotentiate LTP (Barrionuevo et al. 1980; Fujii et al. 1991; Wagner and Alger 1996). LTP was induced in one of two independent s. radiatum pathways by giving three trains of HFS (100 Hz/1 s) at 20-s intervals to the test pathway (Fig. 2). This protocol produces maximal LTP (e.g., Oliet et al. 1997; Wagner and Alger 1995). The control pathway did not receive HFS. After LTP had been established for 40 min (151.0 ± 4.1% of baseline, n = 10), DHO was applied for 15 min and then washed for 1 h, after which a second HFS was delivered to the test pathway. Figure 2A shows sample traces of responses from the test and control pathways. LTP of the fEPSP (top left) was induced by HFS in the test pathway but not in the control pathway (bottom left). Traces show the effects of DHO (center column) on these responses and the responses (right) after washing DHO from the chamber. The original control traces are reproduced as 
for comparison. Note the persistent reduction of the fEPSP back to control level (top insets) or below (bottom insets) after DHO removal. We confirmed that the potentiated responses were depotentiated, and not nonselectively suppressed by DHO, by demonstrating that if an additional bout of HFS was given after wash of DHO for >50 min, LTP could be reinstated (Fig. 2B, top). Because the initial potentiating protocol will induce maximal LTP, the ability to reinstate LTP after DHO removal demonstrates a specific reversal of the LTP process (Mulkey and Malenka 1992).
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20%) as were the responses in naïve slices (see Fig. 1A). These results suggest that DHO causes a long-term suppression of excitatory synaptic responses that resembles LFS-LTD. DHO-LTD is independent of NMDAR, mGluR or cannabinoid receptor activation
Two forms of LFS-LTD have been identified in the rat hippocampal CA1 region (Oliet et al. 1997). One is mediated by NMDA receptors and the other is mediated by mGluRs. Inhibition of the Na,K pump in presynaptic nerve terminals could cause an increase in intraterminal [Ca2+] followed by release of glutamate and activation of these receptors. To determine if DHO-LTD requires activation of NMDA or mGlu receptors, we applied DHO in the presence of 50–100 µM APV or the mGluR antagonist, LY341495 (100 µM). In APV, DHO suppressed fEPSPs to 87.3 ± 2.4% baseline after 50 min of DHO wash (n = 7, P < 0.05; data not shown). In LY341495, DHO suppressed the mean fEPSPs as much (84.7 ± 4.7% of baseline, n = 3), although because of the greater variability of the baseline responses in LY34195, this was not statistically significant. In the presence of a combination of APV and LY341495, DHO could depotentiate LTP responses to the same extent (i.e., to 62.2 ± 3.9% of the LTP level; n = 3) as in control conditions (data not shown). We confirmed that the drug cocktail was effective by showing that it prevented LTP induction by a second bout of HFS. Together these experiments suggest that DHO mediates a long-term suppression of excitatory synaptic responses and reversal of LTP that is largely independent of NMDA or mGlu receptors.
The endocannabinoids anandamide and 2-arachidonylyglycerol can reduce glutamate release (Al-Hayani and Davies 2000; Alger 2002; for review) although the mechanism is controversial (Hajos et al. 2001). Endocannabinoids can be released by a variety of endogenous processes (Alger 2002). To test the possibility that endocannabinoids are involved in DHO-LTD, we bathed slices with saline containing the selective CB1 receptor antagonist AM-251 (4 µM) for 20 min prior to the standard DHO application. We also assessed maximal DHO suppression at 5 min after starting the wash, and the point of half-maximal suppression caused by DHO, which occurs at 8 min after beginning DHO application. AM-251 did not prevent DHO-induced suppression at 5 min of wash (54.1 ± 0.8% of baseline, n = 6, P < 0.001, data not shown) or at 25 min of wash (74.4 ± 0.2% of baseline, n = 6, P < 0.001). We did notice that it was not until the 12th minute of DHO application that half-maximal suppression was observed (81.2 ± 0.5% of baseline, P < 0.001). Inasmuch as DHO-induced suppression is usually significant after only 8 min of DHO application, this suggests that AM-251 causes a delay in the onset of the DHO effect, and indeed this is quantitated in Fig. 5A. Thus the initial suppression, although not DHO-LTD, may be mediated in part by cannabinoid receptors.
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Thus far the data show that blocking the Na,K-pump did not cause DHO-LTD by liberating some likely extracellular receptor ligands. We next considered the possibility that pump inhibition could cause its effects by activating intracellular effectors downstream of extracellular ligands: e.g., protein kinases. The p38 MAP kinase-signaling pathway has been implicated in the induction and maintenance of mGluR-LTD at both the CA3-CA1 (Bolshakov et al. 2000) and the medial perforant path-dentate gyrus synapse (Rush et al. 2002) in the rat hippocampus. To discover if p38 MAP kinase was involved in DHO-LTD, we preincubated hippocampal slices with the selective p38 MAP kinase inhibitor, SB 203580 (25 µM) for 1 h prior to DHO application. We found SB 203580 failed to prevent DHO-induced suppression at 5 min (60.5 ± 1.0% of baseline, n = 7, P < 0.01) or at 25 min of wash from DHO (68.1 ± 0.6% of baseline, n = 7, P < 0.01). Interestingly, SB 203580 delayed the onset of suppression (81.96 ± 1.02 of baseline at 11 min of DHO) as did AM-251, showing that the drug did have an effect (Fig. 5A).
Protein kinase C (PKC) has been implicated in the induction of mGluR-dependent LTD in the rat dentate gyrus, where the PKC-selective inhibitor bisindoylmaleinmide I (GF 109 203X; BIS-1) blocked group I mGluR-mediated induction and expression of LTD (Camodeca et al. 1999). Nevertheless, preincubating slices with BIS-1 (5 µM) for 1 h before the application of DHO failed to prevent either the induction (46.9 ± 2.6% of baseline, n = 7, P < 0.001, DHO-8 min; 29.4 ± 1.3% of baseline, n = 7, P < 0.001, 5-min wash) or expression of DHO LTD (53.9 ± 0.4% of baseline, n = 7, P < 0.001). It appears that neither of two prominent intracellular cascades implicated in synaptic LTD mediates DHO-LTD.
LFS-LTD and DHO-LTD do not co-occlude
The previous results suggested that, despite phenomenological similarities, LFS-LTD and DHO-LTD might not be caused by the same mechanisms. To obtain direct evidence on this point, we induced first one and then the other form of LTD in a given group of slices. If both forms shared the same mechanisms, then they should co-occlude. We found no evidence for co-occlusion, however (Fig. 3). When DHO-LTD was induced first and allowed to stabilize for 25 min after removing DHO, three subsequent bouts of LFS given at 10-min intervals (each bout consisting of 900 pulses at 1 Hz), produced LFS-LTD of 25% (as measured from the final DHO-LTD amplitude). That is, DHO-LTD produced at stable depression of 20% from control amplitudes, and LFS-LTD a further 25% reduction from that level (Fig. 3A, n = 5). Conversely, when saturated LFS-LTD was induced first with three bouts of stimulation, application of DHO for 15 min induced a further stable depression from that level (Fig. 3B, n = 5). In other words, each induction method caused about the same degree of depression whether it was delivered to naïve, or to already depressed, slices. Hence LFS-LTD and DHO-LTD appear to represent distinct phenomena.
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Recently, Hu et al. (2001) discovered that BK-type, calcium-activated, K+ channels in the presynaptic terminals of glutamatergic synapses in the rat hippocampus can regulate glutamate release when basal levels of release are elevated, although they do not do so under normal conditions. Subsequently this group found (Gu and Storm 2002) that experimental hypoxia/ischemia caused a reduction in fEPSPs that was blocked by the BK channel inhibitor iberiotoxin (IbTX). One possibility is that the rise in [Ca2+]i caused by hypoxia/ischemia led to a increase in BK channel activity in presynaptic nerve terminals that shunted the presynaptic action potential and decreased the probability of transmitter release. Because a major consequence of hypoxia/ischemia is a reduction in Na,K pump activity, we considered the possibility that presynaptic BK channels would affect glutamate release during DHO treatment.
To reduce the quantities of IbTX required for these experiments, we assessed the kinetics and magnitude of the DHO effect, and DHO-LTD at 25 min of wash from DHO. We began by bath-applying IbTX (100 nM) for 10 min prior to, during, and for 25 min after DHO application. IbTX had no effect on baseline transmission, as reported (Hu et al. 2001). Remarkably, however, IbTX completely abolished DHO-LTD (compare Fig. 4, A and B). At 25 min of wash, the fEPSP was 96.2 ± 0.7% of baseline, n = 7, P < 0.01). Moreover, IbTX prevented the initial suppression that normally occurs during the first 8 min of DHO application (99.9 ± 0.5% of baseline, n = 7), and reduced the depression of the fEPSP seen at 5 min of wash, although the remaining depression was still significant (81.4 ± 3.5% of baseline, P < 0.001).
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Comparison of the results in Fig. 4, B and C, suggests that the presence of IbTX during the wash period is necessary to prevent DHO-LTD expression, but IbTX applied only during the wash is insufficient for DHO-LTD prevention. When the application of IbTX coincided with the beginning of the saline wash, it did not reverse the synaptic depression (68.3 ± 0.2% of baseline, n = 3, P < 0.001, 25-min wash, Fig. 4D). We conclude that IbTX had to be present during DHO application and for 10–15 min after starting the wash to block the induction or expression of DHO-LTD.
We predicted that application of the broader spectrum K+ blocker, TEA, would mimic IbTX. TEA, applied at 2 mM, a concentration that blocks BK channels in hippocampal cells (Lancaster and Nicoll 1987) throughout the entire recording period significantly reduced the suppression at 25-min wash (86.3 ± 1.1% of baseline, n = 6, P > 0.1, data not shown) but did not prevent the initial DHO-induced suppression (to 62.2 ± 1.1% of baseline, n = 6, P < 0.001 at 8-min DHO; to 49.8 ± 2.0% of baseline at 5-min wash, P < 0.001).
Time course of DHO-LTD: a between-groups analysis
To compare the effects of the various pharmacological manipulations with the control experiments (DHO-only) and each other across the three measured time points, we performed a two-way repeated-measures ANOVA on the group means followed by a Scheffe post hoc analysis. The Scheffe test is the most conservative test for multiple comparisons, maintaining the set alpha value for all comparisons ( = 0.01). The results of the analysis, which are subdivided by the different time points, are illustrated in Fig. 5.
From Fig. 5A, it can be seen that IbTX, AM-251 and SB 203580 delayed the onset of depression after DHO application. That there was no difference among these three groups suggests the possibility of converging signal pathways. Although we have no direct evidence for this, CB1 receptors have been linked to the activation of the p38 MAP kinase pathway (Bouaboula et al. 1995). BIS-1 slightly decreased the time of onset of DHO-induced depression. Only IbTX (when it was present throughout) reduced the amount of depression seen at the 5-min wash point (Fig. 5B). DHO-LTD was largely prevented by continuous application of IbTX and TEA (Fig. 5C).
Finally, we addressed the question of whether or not the BK channels had to be activated by action potentials during the DHO application for DHO-LTD to be expressed. Normally we delivered field potential stimuli at 20-s intervals throughout the DHO application period, and the action potentials in the terminals would have caused the opening of these channels. To explore the issue, we carried out two-pathway experiments (n = 5) in which stimulation was continued in one pathway (S1), but suspended in the other (S2), throughout DHO application period and for 20 min into the wash (Fig. 6A). We then resumed 0.05-Hz stimulation in the nonstimulated pathway. The fEPSPs in both pathways were significantly different from their respective control values (S1: to 76.6 ± 1.96% of control; S2: to 82.7 ± 1.85% of control) and not different from each other. DHO-LTD did not require periodic action potential activation of BK channels for its induction or expression. We confirmed that the same mechanism of DHO-LTD was operant whether or not the stimulation was given, by applying IbTX to a separate set of slices (n = 5) also studied with the two-pathway protocol (Fig. 6B). Continuous application of IbTX (until 15 after starting DHO wash) prevented DHO-LTD in (S1: to 95 ± 0.96% of control) and unstimulated (S2: to 95 ± 0.86% of control) pathways. A two-way, repeated-measures ANOVA showed that in IbTX neither S1 nor S2 responses differed from their control values and that both differed from the responses at comparable time points in the absence of IbTX.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) and release neurotransmitters or neuromodulators. An obvious mechanism for the present experiments was glutamate release. However, unlike the two forms of synaptic LTD in CA1 (Oliet et al. 1997) and depotentiation by LFS-LTD (Wagner and Alger 1996), DHO-induced reversal of LTP appears to be largely independent of NMDA or mGluR activation. Alternatively, endocannabinoids released as a result of synaptic activity can induce LTD (Gerdeman et al. 2002), but an antagonist of the CB1 receptor did not prevent DHO-LTD. Finally, there was no evidence of co-occlusion between LFS-LTD and DHO-LTD. We conclude that, although DHO-LTD and LFS-LTD resemble each other phenomenologically, they do not depend on the same cellular mechanisms.
Na,K-pump inhibition can activate signaling pathways downstream of surface receptors. When bound by ouabain, Na+,K+-ATPase triggers Ras-dependent signaling pathways in rat cardiac myocytes and essentially acts as a signal transduction protein (Liu et al. 2000; Mohammadi et al. 2001). We considered p38 MAP kinase and PKC as potential downstream mediators of DHO-LTD but did not affect expression of DHO-LTD by applying inhibitors of these enzymes. BIS-1 did not block the induction of DHO-LTD, in contrast to its effects on mGluR-LTD (Camodeca et al. 1999).
Analysis of the time course of DHO-LTD induction and expression revealed that the CB1 receptor antagonist, AM-251 and the p38 MAP kinase inhibitor, SB 203580, both delayed the onset of fEPSP suppression. Thus endocannabinoids could mediate the initial effects of DHO, although one caveat is that AM-251 is an inverse agonist and can, by sequestering G proteins, affect other G-protein-coupled receptors (Vasquez and Lewis 1999). Inhibition of p38 MAP kinase blocks induction of mGluR-LTD (Bolshakov et al. 2000) and has been linked to CB1 receptor activation (Bouaboula et al. 1995), so it is possible that the similarity of effects of AM-251 and SB 203580 on DHO actions reflect inhibition of the same signaling cascade.
Previous work (McCarren and Alger 1987; Vaillend et al. 2002) showed that low concentrations of DHO had no persistent effects on membrane properties or on extracellular ion concentrations, suggesting that the postsynaptic cells are not strongly affected. However, DHO at low concentrations also does have subtle postsynaptic effects. For instance, it enhances the activation of a voltage-dependent Ca2+ conductance (McCarren and Alger 1987) and suppresses NMDA receptor-dependent responses to a greater extent than it does AMPA receptor-dependent responses (Vaillend et al. 2002). In CA1 cells, NMDA-dependent LTP is largely a postsynaptic phenomenon (Malenka and Nicoll 1999), and DHO would therefore have to act postsynaptically to cause depotentiation. Nevertheless, we have no direct evidence for the actual locus of DHO-LTD expression or induction or of DHO-depotentiation. Future work will be required to address these issues.
A possible role for presynaptic increases in [Ca2+]i in DHO-LTD is suggested by the effects of the BK channel blocker IbTX on DHO-induced suppression of fEPSPs. A combined immunohistochemical and EM study revealed that BK channels are concentrated at glutamatergic nerve terminals in CA1 (Hu et al. 2001). Interestingly Hu et al. found that these channels do not normally take part in repolarizing the terminal action potential. When the presynaptic action potentials are broadened by 100 µM 4-AP, which blocks other K channels, Ca2+ enters the terminals, BK-type channels are activated, and transmitter release is reduced. Under these conditions, IbTX enhances glutamate release. Our results are compatible with the speculation of Hu et al. (2001) that BK channels would be activated under hypoxic or ischemic conditions because of an increase in intraterminal [Ca2+] and would perhaps have some protective role. Increases in [Ca2+]i would activate BK channels, speeding repolarization of the terminals and reducing glutamate release. On the other hand, the residual DHO-induced fEPSP suppression in IbTX was similar in magnitude and time of peak effect to the DHO-induced suppression of the presynaptic fiber volley in control conditions (compare Figs. 1B and 4B), which suggests that DHO may differentially affect the various ion channels that modulate fiber conduction and the release process. This idea is compatible with the observations of Hu et al. (2001) that IbTX affected transmitter release but not the shape of the presynaptic fiber volley. It is also compatible with the complex, biphasic time course of fEPSP suppression that we often observed (e.g., Fig. 1). The initial phase would represent suppression of the release mechanism at the nerve terminal, which begins to reverse soon after DHO removal, but which is interrupted by the secondary phase of fEPSP suppression that coincides with the delayed suppression of the fiber volley.
Understanding the prevention of DHO-LTD by continuous application of IbTX is more difficult. When IbTX was present only when DHO was being applied, it did not alter DHO-LTD induction. When IbTX application began as DHO was being washed from the chamber, it had no effect, which showed that persistent activation of BK channels cannot explain DHO-LTD expression. To prevent DHO-LTD, IbTX had be present for an interval that extended from near the end of the DHO treatment period for 10–15 min into the wash period. Evidently intracellular events initiated during this period by partial inhibition of the Na,K-pumps are largely responsible for DHO-LTD induction. The final interpretation of the data depends on the actual loci of DHO-LTD induction and the relevant IbTX-sensitive channels. Despite their prevalence on presynaptic terminals, such channels do exist on CA1 somata, and therefore a postsynaptic site of DHO-LTD prevention by IbTX is conceivable.
This study highlights the effects of partial Na,K-pump inhibition on neuronal activity and thereby indirectly provides evidence for a physiological or pathophysiological role for endogenous Na,K-pump inhibitors. Although we focused on different neurophysiological endpoints than did Ross and Soltesz (2000, 2001), our data are compatible with theirs in suggesting the possibility of long-term synaptic plasticity associated with Na,K-pump dysfunction. Our findings might be relevant for evaluating the role of endogenous ouabain- or DHO-like compounds (Hamlyn et al. 1991; Qazzaz et al. 2000) or other Na,K-pump modulators (Therien et al. 2000) in regulating neuronal synaptic plasticity.
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ACKNOWLEDGMENTS |
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This work was funded by National Institutes of Neurological Disorders and Stroke Grants NS-36612 and NS-30219 to B. E. Alger. Drs. C. Reich and S. Mason were supported by the Cellular and Integrative Neuroscience Postdoctoral Training Grant (NS-07375) awarded to the University of Maryland School of Medicine.
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FOOTNOTES |
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* C. G. Reich and S. E. Mason contributed equally to this work. 
Address for reprint requests and other correspondence: B. E. Alger, Dept. Physiol., Univ. MD Sch. Med., 655 W. Baltimore St., Baltimore, MD 21201 (E-mail: balger{at}umaryland.edu).
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Al-Hayani A and Davies SN. Cannabinoid receptor mediated inhibition of excitatory synaptic transmission in the rat hippocampal slice is developmentally regulated. Br J Pharmacol 131: 663–665, 2000.[CrossRef][ISI][Medline]
Beattie EC, Carroll RC, Yu X, Morishita W, Yasuda H, von Zastrow M, and Malenka RC. Regulation of AMPA receptor endocytosis by a signaling mechanism shared with LTD. Nature Neurosci 3: 1291–1300, 2000.[CrossRef][ISI][Medline]
Berrebi-Bertrand I, Maixent J-M, Christe G, and Lelièvre LG. Two active Na+/K+-ATPases of high affinity for ouabain in adult rat brain membranes. Biochem Biophys Acta 1021: 148–156, 1990.[Medline]
Barrionuevo G, Schottler F, and Lynch G. The effects of repetitive low frequency stimulation on control and "potentiated" synaptic responses in the hippocampus. Life Sci 27: 2385–2391, 1980.[CrossRef][ISI][Medline]
Bear MF and Abraham WC. Long-term depression in hippocampus. Annu Rev Neurosci 19: 437–462, 1996.[CrossRef][ISI][Medline]
Blaustein MP. Physiological effects of endogenous ouabain: control of intracellular Ca2+ stores and cell responsiveness. Am J Physiol Cell Physiol 264: C1367–C1387, 1993.
Bolshakov VY, Carboni L, Cobb MH, Siegelbaum SA, and Belardetti F. Dual MAP kinase pathways mediate opposing forms of long-term plasticity at CA3-CA1 synapses. Nat Neurosci 3: 1107–1112, 2000.[CrossRef][ISI][Medline]
Bouaboula M, Poinot-Chazel C, Bourrie B, Canat X, Calandra B, Rinaldi-Carmona M, Le Fur G, and Casellas P. Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1. Biochem J 312: 637–641, 1995.[ISI][Medline]
Camodeca N, Breakwell NA, Rowan MJ, and Anwyl R. Induction of LTD by activation of group I mGluR in the dentate gyrus in vitro. Neuropharmacology 38: 1597–1606, 1999.[CrossRef][ISI][Medline]
Dudek SM and Bear MF. Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-D-aspartate receptor blockade. Proc Natl Acad Sci USA 89: 4363–4367, 1992.
Fitzjohn SM, Kingston AE, Lodge D, and Collingridge GL. DHPG-induced LTD in area CA1 of juvenile rat hippocampus; characterisation and sensitivity to novel mGlu receptor antagonists. Neuropharmacology 38: 1577–1583, 1999.[CrossRef][ISI][Medline]
Fujii S, Saito K, Miyakawa H, Ito K, and Kato H. Reversal of long-term potentiation (depotentiation) induced by tetanus stimulation of the input to CA1 neurons of guinea pig hippocampal slices. Brain Res 555: 112–122, 1991.[CrossRef][ISI][Medline]
Gerdeman GL, Ronesi J, and Lovinger DM. Postsynaptic endocannabinoid release is critical to long-term depression in the striatum. Nat Neurosci 5: 446–451, 2002.[ISI][Medline]
Gu N and Storm JF. Regulation of glutamate release by presynaptic calcium-activated potassium channels (BK channels) is enhanced under hypoxic conditions in rat hippocampus (CA1). Soc Neurosci Abstr 32: 340, 2002.
Haddad GG and Jiang C. O2 deprivation in the central nervous system: on mechanisms of neuronal response, differential sensitivity and injury. Prog Neurobiol 40: 277–318, 1993.[CrossRef][ISI][Medline]
Hajos N, Ledent C, and Freund TF. Novel cannabinoid-sensitive receptor mediates inhibition of glutamatergic synaptic transmission in the hippocampus. Neuroscience 106: 1–4, 2001.[CrossRef][ISI][Medline]
Hamlyn JM, Blaustein MP, Bova S, DuCharme DW, Harris DW, Mandel F, Mathews WR, and Ludens JH. Identification and characterization of a ouabain-like compound from human plasma. Proc Natl Acad Sci USA 88: 6259–6263, 1991.
Hu H, Shao LR, Chavoshy S, Gu N, Trieb M, Behrens R, Laake P, Pongs O, Knaus HG, Ottersen OP, and Storm JF. Presynaptic Ca2+-activated K+ channels in glutamatergic hippocampal terminals and their role in spike repolarization and regulation of transmitter release. J Neurosci 21: 9585–9597, 2001.
Huber KM, Kayser MS, and Bear MF. Role for rapid dendritic protein synthesis in hippocampal mGluR-dependent long-term depression. Science 288: 1254–1256, 2000.
Kamal A, Ramakers GMJ, Urban IJA, De Graan PNE, and Gispen WH. Chemical LTD in the CA1 field of the hippocampus from young and mature rats. Eur J Neurosci 11: 3512–3516, 1999.[CrossRef][ISI][Medline]
Kemp N, McQueen J, Faulkes S, and Bashir ZI. Different forms of LTD in the CA1 region of the hippocampus: role of age and stimulus protocol. Eur J Neurosci 12: 360–366, 2000.[CrossRef][ISI][Medline]
Lancaster B and Nicoll RA. Properties of two calcium-activated hyperpolarizations in rat hippocampal neurones. J Physiol (Lond) 389: 187–203, 1987.
Lee H-K, Kameyama K, Huganir RL, and Bear MF. NMDA induces long-term synaptic depression and dephosphorylation of the GluR1 subunit of AMPA receptors in hippocampus. Neuron 21: 1151–1162, 1998.[CrossRef][ISI][Medline]
Lees GJ. Inhibition of sodium-potassium-ATPase: a potentially ubiquitous mechanism contributing to central nervous system neuropathology. Brain Res Rev 16: 283–300, 1991.[CrossRef][Medline]
Linden DJ. Long-term synaptic depression in the mammalian brain. Neuron 12: 457–472, 1994.[CrossRef][ISI][Medline]
Liu J, Tian J, Haas M, Shapiro JI, Askari A, and Xie Z. Ouabain interaction with cardiac Na+/K+-ATPase initiates signal cascades independent of changes in intracellular Na+ and Ca2+ concentrations. J Biol Chem 275: 27838–27844, 2000.
Malenka RC and Nicoll RA Long-term potentiation—a decade of progress? Science 285: 1870–4.
McCarren M and Alger BE. Sodium-potassium pump inhibitors increase neuronal excitability in the rat hippocampal slice: role of a Ca2+-dependent conductance. J Neurophysiol 57: 496–509, 1987.
McNamara JO. Cellular and molecular basis of epilepsy. J Neurosci 14: 3413–3425, 1994.[ISI][Medline]
Mohammadi K, Kometiani P, Xie Z, and Askari A. Role of protein kinase C in the signal pathways that link Na+/K+-ATPase to ERK1/2. J Biol Chem 276: 42050–42056, 2001.
Morishita W and Alger BE. Direct depolarization and antidromic action potentials transiently suppress dendritic IPSPs in hippocampal CA1 pyramidal cells. J Neurophysiol 85: 480–484, 2001.
Mulkey RM, Herron CE, and Malenka RC. An essential role for protein phosphatases in hippocampal long-term depression. Science 261: 1051–1055, 1993.
Mulkey RM and Malenka RC. Mechanisms underlying induction of homosynaptic long-term depression in area CA1 of the hippocampus. Neuron 9: 967–975, 1992.[CrossRef][ISI][Medline]
Nicoll RA and Alger BE. A simple chamber for recording from submerged brain slices. J Neurosci Methods 4: 153–156, 1981.[CrossRef][ISI][Medline]
Oliet SHR, Malenka RC, and Nicoll RA. Two distinct forms of long-term depression coexist in CA1 hippocamal pyramidal cells. Neuron 18: 969–982, 1997.[CrossRef][ISI][Medline]
Qazzaz HM, El Masri MA, and Valdes R Jr. Secretion of a lactonehydrogenated ouabain-like effector of sodium, potassium-adenosine triphosphatase activity by adrenal cells. Endocrinology 141: 3200–3209, 2000.
Ross ST and Soltesz I. Selective depolarization of interneurons in the early posttraumatic dentate gyrus: involvement of the Na+/K+-ATPase. J Neurophysiol 83: 2916–2930, 2000.
Ross ST and Soltesz I. Long-term plasticity in interneurons of the dentate gyrus. Proc Natl Acad Sci USA 98: 8874–8879, 2001.
Rush AM, Wu J, Rowan MJ, and Anwyl R. Group I metabotropic glutamate receptor (mGluR)-dependent long-term depression mediated via p38 mitogen-activated protein kinase is inhibited by previous high-frequency stimulation and activation of mGluRs and protein kinase C in the rat dentate gyrus in vitro. J Neurosci 22: 6121–6128, 2002.
Snyder EM, Philpot BD, Huber KM, Dong X, Fallon JR, and Bear MF. Internalization of ionotropic glutamate receptors in response to mGluR activation. Nat Neurosci 4: 1079–1085, 2001.[CrossRef][ISI][Medline]
Sweadner KJ. Isozymes of the Na+/K+-ATPase. Biochim Biophys Acta 988: 185–220, 1989.[Medline]
Therien AG and Blostein R. Mechanisms of sodium pump regulation. Am J Physiol Cell Physiol 279: C541–C566, 2000.
Vaillend C, Mason SE, Cuttle MF, and Alger BE. Mechanisms of neuronal hyperexcitability caused by partial inhibition of Na+-K+-ATPases in the rat CA1 hippocampal region. J Neurophysiol 88: 2963–2978, 2002.
Vasquez C and Lewis DL. The CB1 cannabinoid receptor can sequester G-proteins, making them unavailable to couple to other receptors. J Neurosci 19: 9271–9280, 1999.
Wagner JJ and Alger BE. GABAergic and developmental influences on homosynaptic LTD and depotentiation in rat hippocampus. J Neurosci 15: 1577–1586, 1995.[Abstract]
Wagner JJ and Alger BE. Homosynaptic LTD and depotentiation: do they differ in name only? Hippocampus 6: 24–29, 1996.[CrossRef][ISI][Medline]
Zho WM, You JL, Huang CC, and Hsu KS. The group I metabotropic glutamate receptor agonist (S)-3,5,-dihydroxyphenylglycine induces a novel form of depotentiation in the CA1 region of the hippocampus. J Neurosci 22: 8838–8849, 2002.
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, HFS trains; 
, the 15-min DHO application. The letters (a–d) indicate the time points at which data were collected for comparison in C (a: control; b: LTP; c: DHO; d: DHO wash). Note that a prior application of DHO does not prevent a 2nd HFS train from inducing LTP in the test pathway (n = 6). C: fEPSP data from the time points in B normalized to the potentiated response at b for each pathway. DHO-induced suppression of the fEPSP slope of the test pathway during the bursting period and after 60 min of DHO wash was not significantly different from the preLTP control (n = 10). In contrast, fEPSP initial slope in the control pathway was significantly suppressed after 60 min of DHO wash (n = 6, P < 0.05).
