Early Maintenance of Hippocampal Mossy Fiber—Long-Term Potentiation Depends on Protein and RNA Synthesis and Presynaptic Granule Cell Integrity

Early maintenance of MF LTP depends on protein synthesis

To determine whether the early maintenance phase of MF LTP depends on synthesis of new proteins, we tested the effect of either one of two powerful protein synthesis inhibitors on recently established MF LTP. To that end, we first induced MF LTP and then, 60 min after HFS, we bath applied either emetine (20 μm; n = 3) or cycloheximide (60 μm; n = 5), each for 60 min. Either inhibitor caused a marked reduction in MF fEPSP amplitude from the enhanced level observed during the first hour after HFS [M_LTP before emetine = 176 ± 8% (Fig. 1B); M_LTP before cycloheximide = 169 ± 7% (Fig. 1C)]. Thirty minutes after onset of either cycloheximide or emetine application, synaptic response amplitudes no longer differed significantly from baseline values [emetine: 127 ± 13% (Fig. 1B); cycloheximide: 129 ± 11% (Fig. 1C); p > 0.3]. This decrease in fEPSP amplitude persisted for >2 hr after washout of emetine or cycloheximide (Fig. 1B,C; Table 1).

To determine whether protein synthesis is involved in the early maintenance of NMDA receptor-dependent LTP in area CA3 as well, we repeated the same experiment except that MK-801 or d-AP5 was omitted from the perfusion bath, and LTP was induced at the commissural—associational input to CA3 pyramidal cells (C/A-LTP). In contrast to the effect of emetine on MF LTP, we found that emetine did not affect C/A-LTP. The amplitude of C/A fEPSPs at the end of 60 min of emetine application (20 μm; 210 ± 15%; n = 3) did not differ significantly from the control pre-drug level (227 ± 11%; 60 min after HFS; p ≤ 0.1) (Fig. 1D; Table 1). Taken together, these findings show that in area CA3, MF LTP but not C/A-LTP requires protein synthesis within 1–2 hr of its induction.

The foregoing results raised the question whether protein synthesis is critical to MF LTP maintenance immediately after its induction. To test this hypothesis, we bath applied emetine (20 μm) for 60 min before delivery of HFS. Emetine did not affect significantly baseline synaptic transmission (108 ± 3% of pre-drug-baseline; n = 3) or PPF (179 ± 20%). Emetine also did not affect MF-PTP (M_PTP = 393 ± 47%; τ_PTP = 2.9 min). However, after the decay of PTP, the amplitude of the fEPSP failed to remain potentiated and rapidly decreased to baseline level (τ_bsln = 9 min) (Figs. ​(Figs.2A,2A, ​,3;3; Table 2). Within 15 min after HFS the amplitude of MF fEPSPs no longer differed significantly from baseline values (120 ± 13%; p > 0.1). A similarly rapid decay time constant of MF LTP (τ_bsln = 12 min) was observed when HFS was delivered in the presence of emetine, i.e., when the 60 min drug application was initiated 30 min before delivery of HFS (n = 3; data not shown). In these experiments, PTP amplitude and decay were comparable with these PTP parameters in control slices (M_PTP = 490 ± 52%; τ_PTP = 3.0 min), similar to the effect that we observed when emetine application was terminated before HFS. Collectively, these data show that recently established MF LTP is strongly dependent on protein synthesis.

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Figure 2.

Early maintenance of MF LTP is blocked by inhibition of either protein or RNA synthesis as well as by MF transection. Group data (mean ± SEM) of the amplitude of MF fEPSPs before and after HFS (upward arrow). The horizontal bar in A, B, and D indicates the duration of drug application before HFS. A, Effect of emetine incubation for 60 min before delivery of HFS to the MF (20 μm; n = 3; triangles). B, Effect of actinomycin-D incubation for 120 min before delivery of HFS to the MF (25 μm; n = 4; filled squares). C, Effect of MF transection performed 120 min before the onset of electrophysiological recordings (n = 5; filled pentagon). The same HFS as delivered in these experiments induced persistent MF LTP in control slices (filled circles; n = 5). D, Joint effect of emetine incubation (20 μm; 60 min before delivery of HFS) and MF transection on MF LTP (n = 4; open squares). Representative traces of MF fEPSPs were obtained 10 min before and 60 min after HFS or 90 min after delivery of HFS in slices with MF transection (asterisk). Calibration: 0.2 mV, 10 msec.

Early maintenance of MF LTP depends on new RNA synthesis

Our findings that protein synthesis is critical to the maintenance of MF LTP not long after its induction prompted us to investigate whether synthesis of a new message also plays a role in the early maintenance phase of MF LTP. To address this issue, we bath applied the RNA synthesis inhibitor actinomycin-D (25 μm) for 120 min before delivery of HFS to MF. Actinomycin-D had no effect on basal synaptic transmission (111 ± 2% of pre-drug baseline; PPF = 160 ± 12%; n = 4), PTP magnitude (M_PTP = 531 ± 61%), or PTP decay (τ_PTP = 2.5 min). However, the potentiation failed to persist, and MF fEPSP amplitudes returned to baseline level with a τ_bsln = 33 min (Fig. 3; Table 2). As suggested by the greater decay time constant for these experiments compared with the rapid decay time constant observed after emetine application, MF responses were still potentiated 15 min after HFS after actinomycin-D application (170 ± 16%; compared with baseline level; p ≤ 0.03). However, within 45 min after HFS the amplitude of MF fEPSPs no longer differed significantly from baseline values (125 ± 10%; p > 0.1) (Fig. 2B). In slices treated with vehicle solution (methanol), PTP and LTP were similar to those measured in control slices (M_PTP = 491 ± 39%; M_LTP = 168 ± 19%; τ_PTP = 2.9 min; n = 3). These data show that the early maintenance phase of MF LTP requires the synthesis of RNA, in addition to the synthesis of proteins.

Early maintenance of MF LTP depends on granule cell soma

Our findings that RNA synthesis is critical for the maintenance of MF LTP not long after its induction raised the question of whether granule cell somas contribute to the early maintenance phase of MF LTP. To address this question, we transected the MF axons near the granule cell bodies >2 hr before HFS. Transection of MF axons had no effect on baseline MF fEPSPs monitored for 90 min (mean amplitude = 0.21 ± 0.04 mV; PPF = 140 ± 21%; n = 5) or on PTP amplitude or decay (M_PTP = 512 ± 41%; τ_PTP = 3.1 min; p > 0.1) (Table 2). However, in the absence of granule cell soma, the potentiated MF fEPSP decayed to baseline level with a time constant comparable with that seen after application of actinomycin-D (τ_bsln = 31 min) (Fig. 3; Table 2). Thus, similar to the effect observed after HFS after actinomycin-D application, the amplitude of MF fEPSPs after MF transection was increased significantly above baseline level 15 min after HFS (196 ± 21%; p < 0.01), but the potentiation did not persist and MF fEPSP amplitudes returned to baseline level within 45 min after HFS (136 ± 19%; p > 0.1) (Fig. 2C). These data indicate that integrity of communication between soma and terminals of the presynaptic granule cell is necessary to maintain MF LTP soon after its induction.

Interestingly, the time course of response decay after HFS in MF transected slices (Fig. 2C) resembled that after HFS after actinomycin-D application (Fig. 2B) but was distinctly slower than that after HFS after emetine application (Fig. 2A). These findings suggest that MF transection and inhibition of RNA synthesis interfere with the same underlying process critical for MF LTP maintenance, whereas inhibition of protein synthesis acts on a different process, the contribution of which is recruited before the one that involves signaling between presynaptic soma and terminals and new RNA synthesis. If this hypothesis of distinct underlying processes is correct, then the time course of response decay after HFS after emetine application should be the same regardless of the intactness of the MF axons. To test this hypothesis, we applied emetine (20 μm) for 60 min before delivery of HFS in slices with MF transected. Emetine had no effect on basal synaptic transmission (109 ± 5% of baseline; PPF = 144 ± 11%; n = 4) or PTP magnitude or decay (M_PTP = 513 ± 98%; τ_PTP = 2.8 min). However, potentiated MF fEPSPs returned to baseline levels with a τ_bsln = 10 min (Figs. ​(Figs.2D,2D, ​,3;3; Table 2). The decay time constant of potentiation in transected slices was similar to that observed after emetine applications in control slices (Figs. ​(Figs.2A,2A, ​,3;3; Table 2). Thirty minutes after HFS, the amplitude of MF fEPSPs no longer differed significantly from baseline values (p > 0.2). These findings corroborate the suggestion that the protein synthesis inhibitor when present before and immediately after LTP-inducing HFS interferes with events unrelated to those affected by inhibition of RNA synthesis or MF transection.

PPF and PTP in slices with transected MF axons were comparable with these measures of presynaptic function in slices with intact MF axons (see above). Nevertheless, it is possible that failure of sustained MF LTP after MF transection is secondary to effects on the function of the postsynaptic CA3 pyramidal cell caused by massive release of glutamate from MF boutons during the transection. To rule out this possibility we tested slices in which the MF axons were transected in the presence of kynurenic acid, a nonselective NMDA, and AMPA/kainate receptor antagonist (10 mm; n = 4). The slices were incubated in kynurenic acid for 60 min after MF transection. The results from kynurenic acid-treated slices were similar to those from slices in which the transection was conducted in the absence of kynurenic acid. The amplitude of MF fEPSPs during baseline was 0.40 ± 0.1 mV (PPF = 160 ± 32%); M_PTP was 403 ± 47%; τ_PTP was 2.8 min; and within 45 min after HFS, the amplitude of the MF EPSPs no longer differed significantly from baseline values (136 ± 19%; data not shown). These control experiments indicate that it is unlikely that a massive release of glutamate after MF transection impacted negatively on subsequent induction and/or maintenance of MF LTP.

It also is possible that the failure of MF LTP maintenance after MF transection is a consequence of a generalized effect on CA3 pyramidal cell function, including its ability to support long-term plasticity. To rule out this possibility, we tested the effect of MF transection on C/A-LTP. In three slices, we monitored both evoked C/A fEPSPs (mean amplitude = 0.22 ± 0.06 mV) (Fig. 4) and evoked MF fEPSPs (mean amplitude = 0.20 ± 0.04 mV). Bath application of the NMDA receptor antagonist d-APV (50 μm) had no effect on evoked MF fEPSPs but caused a slight reduction in C/A fEPSPs (10 ± 2% of pre-drug baseline). Delivery of HFS to the MF input induced robust PTP (M_PTP = 552 ± 45%; τ_PTP = 2.8 min), but the potentiated response decayed to baseline levels within 27 min after HFS (Fig. 4). Thirty minutes after delivery of HFS to the MF input, we delivered a similar HFS to the C/A input. The amplitude of evoked C/A fEPSPs gradually increased (191 ± 17%; 30 min after HFS) and was similar to what we observed in the absence of transected MF (Fig. 1C). The potentiation of the C/A fEPSP was stable for >2 hr and unaffected by bath application of the group II mGluR agonist DCG IV (0.5 m; 169 ± 12% of baseline). In contrast, DCG IV reduced MF fEPSPs below baseline level (67 ± 16%). This selective reduction of MF fEPSPs was reversible. Taken together, these data suggest that MF transection does not affect the ability of CA3 pyramidal cells to maintain LTP induced at a non-MF synaptic input.

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Figure 4.

MF transection does not affect the ability of CA3 pyramidal cells to maintain LTP induced at a non-MF synaptic input. Left panel, MF fEPSPs were recorded in the presence of APV (50 μm) for 20 min before the delivery of HFS (first upward arrow). Delivery of HFS to MF induced PTP but not MF LTP (filled circles; n = 3). Thirty minutes after delivery of HFS to MF, a second HFS delivered to the C/A fibers (second upward arrow) induced stable C/A LTP (open circles). In the presence of DCG IV (0.5 μm for 15 min), MF fEPSPs were reduced selectively. Insert, Schematic of hippocampal slice showing the position of the stimulation and recording electrodes and the transection of MF. Right panel, Single traces of MF fEPSPs (left) and C/A fEPSPs (right). Top traces show fEPSP before and 30 min after delivery of HFS to the C/A input. Only C/A fEPSPs are potentiated (asterisk). Bottom traces show the selective depression of DCG IV on MF fEPSPs(#). The horizontal bar indicates the duration of drug application. Calibration: 0.2 mV, 10 msec.

Early maintenance phase of MF LTP depends on protein and RNA synthesis

Numerous studies have demonstrated that the early maintenance phase of LTP at the MF to CA3 synapse (Frey et al., 1993; Huang et al., 1994; Huang and Kandel, 1996; Nguyen and Kandel, 1996) and the Schaffer collateral to CA1 synapse (Krug et al., 1984; Stanton and Sarvey, 1984; Frey et al., 1988; Huang et al., 1996; Mochida et al., 2001) is independent of protein synthesis. In contrast, other published work indicates that the early maintenance phase of LTP in area CA1 (Deadwyler et al., 1987; Clayton, 2000; Scharf et al., 2002), in the dentate gyrus (Otani and Abraham, 1989; Otani et al., 1989; Nguyen et al., 1994), and at the MF synapse (Barea-Rodríguez et al., 2000) is protein synthesis dependent. In the present study, the inhibitory effect of either emetine or actinomycin-D on the early maintenance of MF LTP cannot be attributed to a drug effect on baseline synaptic transmission or short-term plasticity. Specifically, MF fEPSP amplitudes during baseline were not affected by incubation with either emetine or actinomycin-D. In addition, the time course of amplitude changes of MF fEPSPs after HFS showed that in slices exposed to emetine or actinomycin-D before HFS, the magnitude and decay time constant of PTP were similar to control values and to values reported previously (Langdon et al., 1995; Son and Carpenter 1996). Importantly, MF responses in drug-treated slices remained potentiated well after the decay of PTP. These results indicate that blockade of protein or RNA synthesis selectively inhibited the early maintenance phase of MF LTP without affecting the induction process.

One possible site of action of emetine is the dendrite of the postsynaptic CA3 pyramidal cell. Traditionally, it has been thought that new proteins are synthesized in the cell body of the neuron and transported to the synapse. However, numerous recent studies suggest that train stimulation of afferent fibers induces local protein synthesis in dendrites of the postsynaptic cell (Aakalu et al., 2001;Job and Eberwine, 2001; Smith et al., 2001; Steward and Schuman, 2001; Steward and Worley, 2002). For example, in area CA1 the expression and insertion of AMPA receptors (Kacharmina et al., 2000; Malinow and Malenka, 2002) and the expression of cellular adhesion molecules, such as cadherins and proteins of cytoskeleton (Kim and Lisman 1999; Yamagata et al., 1999; Krucker et al., 2000), are induced in dendritic spines by high-frequency stimulation. It also was shown that the expression of CaM kinase II, a protein critical for the maintenance of LTP (Ouyang et al., 1999; Davis et al., 2000), is induced locally in the dendrites of CA3 pyramidal cells (Chicurel et al., 1993). The time course of protein synthesis in the postsynaptic cell after LTP induction appears to be rapid because new proteins appear within 15–45 min after HFS (Lynch et al., 1994; Osten et al., 1996; Lanahan and Worley, 1998; Ouyang et al., 1999). These data are consistent with the time course of decay of MF LTP in slices treated with emetine described here.

Our data also show that the mechanisms that underlie the maintenance of MF LTP are markedly different from those that underlie the maintenance of LTP at non-MF synapses. Specifically, the early phase of C/A LTP is insensitive to emetine, which indicates that it does not require the synthesis of new proteins. A similar negative effect of emetine was reported for LTP in the CA1 region (Krug et al., 1984; Nguyen et al., 1994). In addition to inducing rapid protein synthesis in dendrites, HFS also induces an increase in the expression of proteins involved in vesicular neurotransmitter release, such as synapsin I, synaptotagmin, and synaptophysin (Lynch et al., 1994; Hicks et al., 1997; Sato et al., 2000). It is therefore possible that the synthesis of RNAs and proteins necessary for the maintenance of MF LTP takes place solely in the presynaptic element (granule cell soma; see below).

Finally, the decay of MF LTP produced by emetine is consistent with a role for both presynaptic and postsynaptic cells in the induction and maintenance of MF LTP, as demonstrated recently by Contractor et al. (2002). These authors have identified a retrograde signaling cascade involving Eph B receptor—ephrin ligand interactions that link postsynaptic calcium influx into CA3 pyramidal cells with increased transmitter release by presynaptic MF boutons. Similar to our results with emetine, blockade of the C terminal of EphB2 receptors produced an early decay of MF LTP with little effect on PTP. However, in light of our findings that the disruption of MF LTP maintenance by emetine is insensitive to MF intactness (Figs. 2 A, D), it is tempting to speculate that protein synthesis required for MF LTP maintenance immediately after LTP induction occurs in postsynaptic elements.

In addition to protein synthesis, our results implicate RNA synthesis in the early maintenance of MF LTP. Actinomicyn-D produced a slower decay of the potentiated response than did emetine (Fig. 2B; Table 1). These data are consistent with previous findings from other areas of the hippocampus indicating that levels of different RNAs generally are not increased until ≥30 min after HFS (Sadile et al., 1995; Morimoto et al., 1998; French et al., 2001; Steward and Worley, 2001).