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INTRODUCTION |
-Amino-3-hydroxy-5-methylisoxazolepropionic acid receptor (AMPAR)-mediated excitatory synaptic transmission in the hippocampus can undergo long-term potentiation (LTP) (Bliss and Lømo 1973). LTP commonly is induced by high-frequency stimulation (HFS) of 50-400 Hz in hippocampal CA1 (Anderson et al. 1977
; Dunwiddie and Lynch 1978
; Schwartzkroin and Lester 1975) and dentate gyrus (Bliss and Lømo 1973; Douglas and Goddard 1975). LTP also can be induced by a pairing-type protocol consisting of conjunction of low-frequency stimulation (LFS) and postsynaptic depolarization in CA1 (Gustaffson et al. 1987
; Kauer et al. 1988
). However, the conjunctive-type stimulation failed to induce LTP in other regions, such as cortex (Sah and Nicoll 1991
) and lateral perforant pathway of the dentate gyrus (Colino and Malenka 1993
) and only has been observed in one set of experiments in the medial perforant pathway (Colino and Malenka 1993
).
AMPA-mediated synaptic excitatory transmission also can undergo a long-term decrease, either as depotentiation (DP) from a previously potentiated level (Barrionuevo et al. 1980
; Staubli and Lynch 1990
) or long-term depression (LTD) from a basal level (Dudek and Bear 1992
; Mulkey and Malenka 1992
). LTD usually is induced by LFS at1-5 Hz (Dudek and Bear 1992
; Mulkey and Malenka 1992
). However, it has recently been shown that LTD also can be induced by a conjunctive pairing technique of LFS and depolarization in the medial perforant pathway-granule cell synapse of the dentate gyrus in adult rats. In this study, pairing 60 afferent stimuli at 1 Hz with steady state depolarization at
40 mV under whole cell patch-clamp conditions induced large (40%) LTD, a similar magnitude to that induced by standard LFS (Wang et al. 1997
).
The induction of both LTP (Lynch et al. 1983
) and LTD (Mulkey and Malenka 1992
) are known to be Ca2+ dependent. The rise in intracellular Ca2+ is known to stimulate kinases, which are essential for LTP induction (Malenka et al. 1989
; Malinow et al. 1989
; Reymann et al. 1988
), and phosphatases, which are essential for LTD induction (Mulkey et al. 1993
). The balance between LTP and LTD induction is believed to be regulated by the differential stimulation of kinases and phosphatases.
The conjunctive pairing-type protocol for the induction of LTD, shown in our previous study (Wang et al. 1997
) was similar to that which previously induced LTP in CA1 (Gustaffson et al. 1987
; Kelso et al. 1986
; Kauer et al. 1988
) except that a milder depolarization was used to induce LTD in our previous study of LTD [to just
40 mV (Wang et al. 1997
)]. In the present study, we have investigated the effects of stimulation with the conjunctive pairing protocol on the induction of LTP and LTD in the medial perforant pathway of the dentate gyrus, in particular using a stronger depolarization than that used previously and also studying the effect of increasing the stimulation frequency.
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METHODS |
All experiments were carried out on transverse slices of the rat hippocampus (weight 40-80 g) as previously described (O'Connor et al. 1995
; Wang et al. 1996
). The brains were removed rapidly after decapitation and placed in cold oxygenated (95%O2-5% CO2) media. Slices were cut at a thickness of 350 µm using a Campden vibroslice, and placed in a holding chamber containing oxygenated media at room temperature. The slices then were transferred as required to a submerged recording chamber and continuously superfused at a rate of 5-8 ml/min at 30-32°C.
The control media contained (mM) 120 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2.0 MgSO4, 2.0 CaCl2, and 10 D-glucose. All solutions contained 100 µM picrotoxin (Sigma) to block
-aminobutyric acid-A-mediated activity. Additional drugs used were ruthenium red (Calbiochem), D(
)-2-amino-5-phosphonopentanoic acid (D-AP5) (Tocris Cookson). The patch-clamp electrode, resistance 5-8 M
, had a standard intracellular solution of (in mM) 130 potassium gluconate, 10 KCl, 10 ethylene glycol-bis(
-aminoethyl ether)-N,N,N
,N
-tetraacetic acid (EGTA), 1 CaCl2, 3 MgCl2, 20 N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES), 5 MgATP, and 0.5 NaGTP, pH 7.2 (using KOH). Such medium was calculated to give a free intracellular Ca2+ concentration of 17.5 nM. A set of experiments also was carried out using an intracellular solution containing 140 potassium gluconate, 10 KCl, 0.2 EGTA, 0.01 CaCl2, 3 MgCl2, 20 HEPES, 5 MgATP, and 0.5 NaGTP. Such medium was calculated to give a free intracellular Ca2+ concentration of ~50 nM. Whole cell recordings from dentate granule cells were made using an Axopatch 1D amplifier (3 kHz low-pass Bessel filter). The capacatitive current was cancelled electronically, and the series resistance (9-18 M
, as measured directly from the amplifier) compensated by 60-70%. The mean input resistance was 249 ± 16 (SE) M
, and the mean resting potential
71 ± 4 mV. The input resistance was monitored continuously, and the recording terminated if it varied by >10%.
Presynaptic stimulation was applied to the medial perforant pathway. Excitatory postsynaptic currents (EPSCs) were recorded at a control frequency of 0.033 Hz and at a holding potential of
70 mV. The amplitude of the test EPSC was adjusted to one-third of maximum, usually 50-100 pA. LTD and LTP were induced as described below. The stimulation voltage during HFS was identical to that used for the test stimulation. Recordings were analyzed using the Strathclyde electrophysiological software (Dr. J. Dempster). Values are means ± SE, and Student's t-test was used for statistical comparison.
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RESULTS |
Pairing of LFS and depolarization of 0 mV does not induce LTP or LTD
Pairing of 1-Hz afferent stimulation (50-100 stimuli) with strong steady state depolarization, at 0 mV, did not induce a significant change in the test EPSCs, with neither LTP or LTD being induced. Thus the test EPSC measuring 108 ± 5%, n = 5, P > 0.05 at 30 min poststimulation (Fig. 1A). Conjunctive pairing of stimulation at 2 Hz (60 pulses) with depolarization to 0 mV also failed to induce a significant LTP or LTD, the EPSCs measuring 110 ± 11%, n = 5,P > 0.05 (Fig. 1B). In addition to these experiments with the standard patch pipette solution containing 10 mM EGTA and 1 mM Ca2+, a set of experiments also was carried out in which the intracellular free Ca2+ in the patch pipette was increased by altering the Ca2+/EGTA ratio (0.01 mM CaCl2, 0.2 mM EGTA). Pairing of 1-Hz afferent stimulation(50-100 stimuli) with steady state depolarization at 0 mV using this internal solution was also not found to induce a significant change in the test EPSC, with neither LTP or LTD being induced (test EPSC measured 99 ± 5%, n = 5, P > 0.05; Fig. 1C).

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| FIG. 1.
Pairing-type protocol at a holding potential of 0 mV does not induce long-term potentiation (LTP) or long-term depression (LTD). A: 1-Hz afferent stimulation (50 stimuli) at a steady state holding potential of 0 mV did not induce a significant change in the amplitude of the test EPSC (108 ± 5% at 30 min postpairing). Pipette solution contained 10 mM ethylene glycol-bis( -aminoethyl ether)-N,N,N ,N -tetraacetic acid (EGTA) and 1 mM CaCl2. B: 2-Hz afferent stimulation (60 stimuli) at a steady state holding potential of 0 mV did not induce a significant change in the amplitude of the test excitatory postsynaptic current (EPSC; 110 ± 11% at 30 min postpairing). Pipette solution contiained 10 mM EGTA and 1 mM CaCl2. C: 1-Hz afferent stimulation (50 stimuli) at a steady state holding potential of 0 mV did not induce a significant change in the amplitude of the test EPSC (108 ± 5% at 30 min postpairing). Pipette solution contained 0.2 mM EGTA and 0.1 mM CaCl2.
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Pairing stimulation in the presence of inhibitors of LTD induction results in induction of LTP
It was postulated that the failure to induce LTP or LTD by the pairing technique of LFS stimulation and depolarization to 0 mV is due to the pairing that results in stimulation of the intracellular mechanisms for the induction of both LTP and LTD, resulting in mutual occlusion. Such a theory was tested by investigating whether application of the pairing of 1 Hz LFS and depolarization to 0 mV in the presence of inhibitors of either LTP or LTD induction resulted in either LTD and LTP induction, respectively, being revealed.
Conjunctive pairing experiments were carried out in the presence of two well-known inhibitors of LTD induction, the phosphatase inhibitor okadaic acid (Mulkey et al. 1993
) and ruthenium red (Wang et al. 1997
). In the presence of okadaic acid (200 µM in the patch electrode), the pairing of 1 Hz (50 pulses) with depolarization at 0 mV was found to induce LTP measuring 142 ± 2%, n = 5, P < 0.005, at 30 min poststimulation (Fig. 2A). Ruthenium red (20 µM in the patch electrode) also resulted in the induction of LTP, which measured 140 ± 4%, n = 5, P < 0.005 (Fig. 2B).

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| FIG. 2.
Pairing-type protocol at 0 mV induces LTP if LTD induction is inhibited. One hertz afferent stimulation (50 stimuli) at a steady state holding potential of 0 mV induced LTP measuring 142 ± 2%, P < 0.005, n = 5, at 30 min postpairing when the inhibitor of LTD induction, the phosphatase inhibitor okadaic acid (200 µM) was included in the patch-clamp electrode. B: 1 Hz afferent stimulation (50 stimuli) at a steady state holding potential of 0 mV induced LTP measuring 140 ± 4%, P < 0.001, n = 5, at 30 min postpairing when the inhibitor of LTD induction, ruthenium red (20 µM), was included in the patch-clamp electrode. C: LTP induced by the conjunctive pairing protocol in the presence of okadaic acid was inhibited by D( )-2-amino-5-phosphonopentanoic acid (D-AP5; 50 µM), the EPSC measuring 103 ± 5% at 30 min postpairing (P > 0.05, n = 5).
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LTP has been reported to be blocked by the N-methyl-D-aspartate receptor (NMDAR)-antagonist D-AP5 in the dentate gyrus in extracellular recordings of field potentials (Colino and Malenka 1993
; Wang et al. 1996
; Wigstrom et al. 1986
). The induction of LTP by the pairing technique in the presence of okadaic acid was also NMDAR-dependent, being blocked by the NMDAR-antagonist D-AP5 (50 µM). Thus the pairing of 1 Hz (50 pulses) with depolarizationof 0 mV in D-AP5 did not induce a significant change inthe amplitude of the EPSCs in the presence of okadaic acid(103 ± 5%, n = 5, P > 0.05; Fig. 2C).
Pairing stimulation in the presence of an inhibitor of LTP induction resulted in the induction of LTD
Conjunctive-pairing-type experiments were carried out in the presence of the known inhibitor of LTP induction, K252a, a CaMKII antagonist (Hashimoto et al. 1991
; Wyllie and Nicoll 1994
). In the presence of K-252a (200 µM in the patch pipette), the pairing of 1 Hz (50 pulses) with depolarization of 0 mV resulted in significant induction of LTD (the EPSC measuring 63 ± 6%, n = 5, P < 0.005) after the pairing, i.e., an LTD of 37% (Fig. 3).

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| FIG. 3.
A pairing type protocol induces LTD if LTP induction is inhibited. A: 1 Hz afferent stimulation (50 stimuli) at a steady state holding potential of 0 mV induced LTD measuring 37 ± 4%, P < 0.001, n = 5, at 30 min postpairing when the inhibitor of LTP induction, the kinase inhibitor K252a (200 µM) was included in the patch pipette.
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Effect of increased frequency of afferent stimulation in cells held at 0 mV
It has been shown previously that LTP of field potentials can be induced by HFS in the medial perforant pathway of the dentate gyrus (Wang et al. 1996
; Wigstrom and Gustaffson 1983
), an LTP that was NMDAR-dependent (Colino and Malenka 1993
; Hanse and Gustafsson 1992). In the present study, an investigation was carried out to determine whether HFS could induce NMDAR-dependent LTP of intracellular EPSCs recorded using whole cell patch clamping in the medial perforant pathway-granule cell synapse.
Applying HFS (1 train of 8 pulses at 200 Hz, intertrain interval 2 s) under voltage-clamp conditions at a holding potential of 0 mV resulted in the induction of LTP measuring 150 ± 6% (P < 0.001, n = 5; Fig. 4A). An LTP of only slightly higher amplitude was induced by HFS of eight trains of eight pulses at 200 Hz (158 ± 5%) applied at a holding potential of 0 mV. The HFS-induced LTP of whole cell EPSCs was NMDAR-dependent, being inhibited by D-AP5. Thus LTP after one train of eight pulses, 200 Hz at 0 mV, was strongly blocked in the presence of D-AP5 (50 µM), measuring 102 ± 3%, P < 0.001, n = 5 (Fig. 4A). HFS-induced LTP also could be induced in cells after the inability of the conjunctive-pairing protocol to induce LTP (Fig. 4B).

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| FIG. 4.
High-frequency stimulation (HFS) applied to the medial perforant pathway induces a N-methyl-D-aspartate receptor (NMDAR)-dependent LTP of EPSCs recorded from granule cells of the dentate gyrus under whole cell patch-clamp conditions. A: , HFS consisting of 1 train of 8 stimuli at 200 Hz, intertrain interval 2.0 s, applied at 0 mV holding potential ( ) induced LTP measuring 150 ± 6% at 30 min post-HFS. , the induction of HFS-induced LTP (1 train of 8 stimuli at 200 Hz, at 0 mV) was blocked by the NMDAR-antagonist, D-AP5 (50 µM), LTP measuring 102 ± 3%. B: example of a cell in which LTP was not induced by the conjunctive pairing of 1-Hz afferent stimulation and steady-state depolarization to 0 mV, but was induced by HFS (1 train of 8 stimuli at 200 Hz).
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DISCUSSION |
Under control conditions, the pairing-type protocol of1-Hz afferent stimulation and strong steady state depolarization of the postsynaptic cell to 0 mV did not induce either LTP or LTD in the medial perforant pathway-granule cell synapse. Such a failure to induce LTP by the pairing-type protocol previously has been observed in pyramidal cells in the anterior cingulate cortex (Sah and Nicoll 1991
) and the lateral perforant pathway-granule-cell synapse (Colino and Malenka 1993
). In fact, the pairing type protocol has been reported only routinely to be successful at inducing LTP in the hippocampal CA1 (Gustafsson et al. 1987; Kauer et al. 1988
), although it has been reported previously to be successful at inducing LTP in a small fraction of cells in the medial perforant pathway in the neocortex (Baranyi and Szente 1987
; Bindman et al. 1988
). Colino and Malenka (1993)
also observed pairing-induced LTP in a small number of cells in the medial perforant path to the dentate gyrus. We were unable to repeat such observations in the present experiments. The relative ease with which LTP can be induced in CA1 by the pairing-type procedure may be related to the very high density of NMDAR in CA1, the highest in the mammalian brain (Monaghan and Cotman 1983
); this results in a very large Ca2+ entry and subsequently a large increase in intracellular Ca2+ concentration, which is particularly effective at inducing LTP rather than LTD (see below).
Experiments in the present study were designed to investigate the mechanisms for the inability of the pairing-type protocol at a holding potential of 0 mV to induce either LTP or LTD. The experiments strongly indicate that the inability of the conjunctive pairing protocol to induce either LTP or LTD in control is due to a mutual occlusion occurring between the stimulation of intracellular kinases, which if produced alone induce LTP, and the stimulation of intracellular phosphatases, which if produced alone would induce LTD. Thus it was found that if stimulation of kinases, and therefore of LTP induction, was prevented with the kinase inhibitor K252a, then the pairing protocol induced LTD, whereas if stimulation of phosphatases was prevented with okadaic acid, then the pairing protocol induced LTP. LTP also was induced if LTD induction was prevented with ruthenium red, an agent previously found to inhibit LTD induction, possibly by preventing release of Ca2+ from intracellular stores (Ma et al. 1988
; Wang et al. 1997
)
The experiments of our previous (Wang et al. 1997
) and present studies demonstrate that the balance between the induction of LTP and LTD by the pairing-type technique in the medial perforant pathway-dentate gyrus synapse depends on two factors: first, the level of steady state depolarization and second, the frequency of afferent stimulation. A lower level depolarization results in the balance between induction of LTP and LTD shifting to the latter state. Thus in a previous study in the dentate gyrus, we have shown that the pairing of 1-Hz afferent stimulation with a mild depolarization to
40 mV induces a large amplitude LTD (Wang et al. 1997
). Previous studies in the visual cortex also have shown that LTD is induced by a weaker depolarization than LTP (Artola et al. 1990
). This shift of the balance between LTP and LTD induction to the latter state with a lower level of depolarization is most likely because weak depolarization results in less Ca2+ influx than strong depolarization and because LTD is induced by a smaller increase in intracellular Ca2+ than LTP. This theory is favored by previous studies showing that the induction of LTD is much less sensitive to a lowering of extracellular Ca2+ than the induction of LTP in the dentate gyrus (Wang et al. 1997
) and in CA1 (Mulkey and Malenka 1992
). Moreover, in experiments involving direct measurement of intracellular Ca2+, LTD was found to be induced by a smaller increase of Ca2+ than LTP in CA1 (Otani and Connor 1996
) and cortex (Yasada and Tsumoto 1996
).
The second factor found to alter the balance between the induction of LTP and LTD was the frequency of the afferent stimulation. Thus increasing the frequency of afferent stimulation from 1 to 200 Hz produced a very dramatic shift toward LTP induction with even eight pulses at 200 Hz inducing large amplitude LTP when the potential was held at 0 mV. Such induction of LTP by 200 Hz may have been produced because of a greater activation of NMDAR at 200 Hz compared with 1 Hz, even with the potential held at 0 mV, which greatly favors activation of NMDAR. Alternatively, the HFS may be increasing the activation of metabotropic glutamate receptors, which are necessary for LTP induction at this synapse (Wang et al. 1995
, 1996
). Metabotropic glutamate receptors are known to be located at perisynaptic sites at hippocampal synapses and therefore will be activated preferentially during HFS due to "spill over" of glutamate (Lujan et al. 1996
).
The induction of LTP in the medial-perforant pathway-dentate gyrus induced by HFS was NMDAR-dependent, verifying previous studies in this pathway (Colino and Malenka 1993
; Hanse and Gustafsson 1992). The induction of LTP by the conjunctive pairing-type protocol of 1-Hz stimulation and steady state depolarization at 0 mV in the presence of the inhibitor of LTD, okadaic acid, also was found to be NMDAR-dependent in the present study. In recent studies (Wang et al. 1997
), it was shown that a NMDAR-independent LTP could be induced by pairing short depolarizing pulses with EPSCs at 1 Hz. The inability to induce a NMDAR-independent LTP by the pairing protocol of steady state depolarization with 1-Hz afferent stimulation is likely to be due to such maintained depolarization producing inactivation of the voltage-gated Ca2+ channels responsible for the NMDAR-independent LTP.