A macromolecular synthesis-dependent late phase of long-term potentiation requiring cAMP in the medial perforant pathway of rat hippocampal slices

Abstract

Memory storage consists of a short-term phase that is independent of new protein synthesis and a long-term phase that requires the synthesis of new proteins and RNA. A cellular representation of these two phases has been demonstrated recently for long-term potentiation (LTP) in both the Schaffer collateral and the mossy fibers of the hippocampus, a structure widely thought to contribute to memory consolidation. By contrast, much less information is available about the medial perforant pathway (MPP), one of the major inputs to the hippocampus. We found that both a short-lasting and a long-lasting potentiation (L-LTP) can be induced in the MPP of rat hippocampal slices by applying repeated tetanization in reduced levels of magnesium. This potentiation was dependent on the activation of NMDA receptors. The early, transient phase of LTP in the MPP did not require either protein or RNA synthesis, and it was independent of protein kinase A activation. By contrast, L-LTP required the synthesis of proteins and RNA, and was selectively blocked by inhibitors of cAMP-dependent protein kinase (PKA). Forskolin, an adenylate cyclase activator, also induced a L-LTP that was attenuated by inhibition of transcription. Our results demonstrate that, like LTP in the Schaffer collateral and mossy fiber pathways, MPP LTP also consists of a late phase that is dependent on protein and RNA synthesis and PKA activity. Thus, cAMP-mediated transcription appears to be a common mechanism for the late form of LTP in all three pathways within the hippocampus.

Fig. 1.

Repeated tetanization induces short- or long-lasting potentiation (L-LTP) in the MPP. A, In the continual presence of 50 μm picrotoxin, two 100 Hz trains (20 sec apart) elicited L-LTP but also induced repetitive afterdischarges after evoked field EPSPs. Sample traces were recorded from a slice at times indicated. Calibration bars: 2 mV, 4 msec.B, Short-lasting potentiation was induced by three 100 Hz trains applied once every minute (at twice test pulse width) starting at “0 min” on the graph. With this protocol, the level of facilitation decayed to <125% of baseline 60–90 min after tetanization. With six trains of 100 Hz stimulation, a longer-lasting potentiation was induced that, although persistent, was variable in magnitude: 3 of 7 slices tested showed <50% facilitation 60 min after tetanization. In both protocols, normal calcium and 50% of normal magnesium levels were present in the saline. C, Ten 100 Hz trains induced a robust and persistent potentiation of MPP field EPSPs when applied in 50% Mg saline (filled circles; n = 11). In contrast, the same protocol elicited only a moderate level of facilitation in normal Mg saline (lower curve, open symbols). The levels of potentiation in normal Mg were significantly lower than those in the reduced Mg saline, beginning 20 min after tetanization and extending to 130 min after tetanization, and averaged only 122% of pretetanus baseline. Sample field EPSP traces were recorded 10 min before and 2 hr after tetanization. Calibration bars: 2 mV, 4 msec. D, Sample field EPSP traces measured from a slice that had been tetanized in reduced Mg saline showed robust potentiation and no repetitive afterdischarges (compare withA). Calibration bars: 2 mV, 4 msec.

Fig. 2.

MPP LTP induced by repeated tetanization is dependent on NMDA receptor activation. The NMDA receptor antagonist APV (100 μm) blocked potentiation when applied for 30 min beginning 20 min before the start of tetanization. The first recorded response after tetanus was obtained 1 min after the last of ten 100 Hz trains (delivered once/min at twice test pulse width); hence, no post-tetanic potentiation was observed. All experiments were performed using saline containing 50% of normal Mg levels.

Fig. 3.

The late phase of LTP in the MPP requires protein synthesis. A1, Potentiation induced by ten 100 Hz trains (delivered once/min at twice test pulse width) was significantly attenuated in slices exposed to 25 μm emetine (a protein synthesis inhibitor) for 40 min. The early phase of LTP, extending from immediately after tetanization until ∼70 min after tetanization, was unaffected by emetine. Sample field EPSP traces were recorded 10 min before and 3 hr after tetanization. Scale bars: 2 mV, 4 msec. A2, Emetine (25 μm) had no effect on short-lasting potentiation induced by three 100 Hz trains (delivered once/min at twice test pulse width). Controls, open symbols; emetine, filled symbols. B, Acute application of 25 μm emetine for 40 min had no marked effects on baseline field EPSPs evoked at 0.02 Hz.

Fig. 4.

Gene transcription mediates the late phase of LTP in the MPP. A1, B1, Two different transcriptional inhibitors, actinomycin D (ACT D; 40 μm) and DRB (0.2 mm), blocked expression of L-LTP when applied for 1 hr, beginning 30 min before start of tetanization. The levels of facilitation were significantly lower in drug-treated slices starting at 80 min after tetanus for ACT D (A1), and at 100 min after tetanus for DRB (B1) (p < 0.05 for unpaired t test comparisons with control slices). Sample field EPSP traces were recorded 10 min before and 3 hr after tetanization in both A1 and B1. Calibration bars for both graphs: 2 mV, 4 msec. A2, B2, Neither 40 μm ACT-D nor 0.2 mm DRB significantly affected baseline field EPSPs evoked at 0.02 Hz. ACT D was dissolved in DMSO (0.1% final concentration).

Fig. 5.

Rp-cAMPS, an inhibitor of PKA, blocks expression of the late phase of LTP in the MPP of hippocampal slices.A1, Slices treated with 50 μmRp-cAMPS for 40 min, beginning 15 min before tetanization, showed decaying LTP that was significantly less than the potentiation observed in untreated control slices 30 min after tetanization and thereafter. Application of Rp-cAMPS overlapped with the tetanization protocol (ten 100 Hz trains, delivered once/min at twice the test pulse width). Sample sweeps were taken 10 min before tetanization and 3 hr after the first tetanus. Calibration bars: 2 mV, 4 msec. A2, Short-lasting potentiation induced by three 100 Hz trains (delivered once/min at twice test pulse width) is unaffected by 50 μm Rp-cAMPS. Rp-cAMPS was applied using the same time window as for the L-LTP graph of part A1. Controls, Open symbols. B, Rp-cAMPS (50 μm) applied for 40 min had no significant effect on baseline field EPSP slopes evoked by 0.02 Hz stimulation of the MPP.

Fig. 6.

A second inhibitor of PKA, KT-5720, also prevents full expression of L-LTP in the MPP. A1, A 30 min application of 1 μm KT-5720, beginning 15 min before tetanization, elicited a delayed decline of potentiation beginning 30–40 min after tetanization. Levels of facilitation in drug-treated slices were significantly lower than those in control slices beginning 60 min after tetanization (p < 0.05 for 60 min after tetanization and all times thereafter). L-LTP was induced using the same protocol as in Figure 5. Sample field EPSP sweeps were recorded 10 min before and 3 hr after the start of tetanization. Calibration bars: 2 mV, 4 msec. A2, Short-lasting facilitation induced by three 100 Hz trains (delivered once/min at twice test pulse width) is unaffected by 1 μm KT-5720. The inhibitor was applied for the same time window as in part A1 of this graph. Controls,Filled symbols. B, Acute exposure of slices to 1 μm KT-5720 for 30 min had no effect on baseline field EPSPs evoked by 0.02 Hz stimulation of the MPP. KT-5720 was dissolved in DMSO (0.1% final concentration).

Fig. 7.

Activation of adenylyl cyclase by forskolin simulates late phase of LTP in the MPP. A, Slices treated with 50 μm forskolin for 15 min showed a gradual potentiation that reached a plateau level ∼45 min after forskolin application. This facilitation persisted for 3 hr during 0.02 Hz test stimulation of the MPP. ACT D (40 μm), a transcription inhibitor, attenuated this facilitation when applied before and during forskolin application. B, In contrast, 50 μm7β-deacetyl-7β-[γ-(morpholino)butyryl]hydrochloride, an inactive forskolin analog, had no effect on baseline field EPSPs evoked at 0.02 Hz. The analog was dissolved in DMSO (0.1% final concentration). Normal magnesium and calcium levels were present in these experiments.