Mitogen-activated protein kinase-mediated reinforcement of hippocampal early long-term depression by the type IV-specific phosphodiesterase inhibitor rolipram and its effect on synaptic tagging

Abstract

Rolipram, a selective inhibitor of cAMP-specific phosphodiesterase 4 (PDE4), has been shown to reinforce an early form of long-term potentiation (LTP) to a long-lasting LTP (late LTP). Furthermore, it was shown that the effects of rolipram-mediated reinforcement of LTP interacts with processes of synaptic tagging (Navakkode et al., 2004). Here we show in CA1 hippocampal slices from adult rats in vitro that rolipram also converted an early form of long-term depression (LTD) that normally decays within 2-3 h, to a long-lasting LTD (late LTD) if rolipram was applied during LTD-induction. Rolipram-reinforced LTD (RLTD) was NMDA receptor- and protein synthesis-dependent. Furthermore, it was dependent on the synergistic coactivation of dopaminergic D(1) and D(5) receptors. This let us speculate that RLTD resembles electrically induced, conventional CA1 late LTD, which is characterized by heterosynaptic processes and synaptic tagging. We therefore asked whether synaptic tagging occurs during RLTD. We found that early LTD in an S1 synaptic input was transformed into late LTD if early LTD was induced in a second independent S2 synaptic pathway during the inhibition of PDE by rolipram, supporting the interaction of processes of synaptic tagging during RLTD. Furthermore, application of PD 98059 (2'-amino-3'-methoxyflavone) or U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), specific inhibitors of mitogen-activated protein kinases (MAPKs), prevented RLTD, suggesting a pivotal role of MAPK activation for RLTD. This MAPK activation was triggered during RLTD by the synergistic interaction of NMDA receptor- and D(1) and D(5) receptor-mediated Rap/B-Raf pathways, but not by the Ras/Raf-1 pathway in adult hippocampal CA1 neurons, as shown by the use of the pathway-specific inhibitors manumycin (Ras/Raf-1) and lethal toxin 82 (Rap/B-Raf).

Figure 1.

Properties of rolipram-reinforced early LTD. A, Transversal hippocampal slice showing the positioning of the electrodes. The two independent S1 and S2 inputs to the same neuronal population and the recording sites for the population spike amplitude and the field EPSP, as well as analog recording traces as representative examples of them, are shown. B, Induction of late LTD in S1 (filled circles) using a repeated LFS protocol (i.e., SLFS; dashed arrow) resulted in late LTD, which is significantly different for the 6 h investigated when compared with a control input S2 (p < 0.05, U test; n = 10). Control stimulation of S2 revealed relatively stable potentials for the time course investigated (open circles). The analog examples given in represent potentials 30 min before (dotted line), 30 min after (dashed line), and 6 h after the induction of the event (here after induction of SLFS in S1; solid line) in input S1 and S2, respectively. Calibration: 3 mV, 3 ms (valid for all single analog examples presented). C, A WLFS protocol (dashed arrow) was used, which elicited transient early LTD of the field EPSP with a duration of 185-210 min (185 min when compared with control input S2; U test; and 210 min when compared with its baseline before WLFS; Wilcoxon test; p < 0.05; n = 7). D, Transformation of early into late LTD by rolipram. Rolipram (0.1 μm) was applied 30 min before WLFS of S1 (filled circles), which normally would induce early LTD (washout of the drug, 30 min after WLFS; open circles, control input; n = 8). E, Same experiment as in D, with the exception that rolipram was applied 25 min after LTD and elapsed for 1 h (n = 7). F, Influence of the NMDA receptor blocker AP-5 on RLTD. After recording a baseline for 50 min, AP-5 (50 μm) was applied for 10 min before being coapplied with rolipram for another 1 h. WLFS in the presence of AP-5 prevented the early LTD (filled circles); the control recording of S2 remained stable during the recorded period (open circles; n = 7). G, Same as in F, but instead of AP-5, the reversible protein synthesis inhibitor anisomycin (25 μm) was applied 10 min before coapplication with rolipram for an additional 1 h (n = 7). H, Effect of a structurally different protein synthesis inhibitor, emetine (20 μm), on RLTD. Analog traces always represent typical field EPSPs 30 min before (dotted line) and 30 min (dashed line) and 6 h (solid line) after WLFS of input S1 (n = 4). Dashed arrows indicate the time point of SLFS or WLFS of the corresponding synaptic input.

Figure 2.

PDE inhibition by rolipram and synaptic tagging. A, Transformation of early into late LTD in S1 (filled circles) by subsequent induction of RLTD in S2 55 min later (open circles; n = 7). WLFS was induced in S1, and 25 min after WLFS of S1, rolipram was added to the bath medium for 1 h. B, Same experiment as in A with the exception of the additional application of D₁ and D₅ receptor antagonist SCH23390 (0.1 μm) 15 min after WLFS of S1 (n = 9). Here, synaptic tagging by RLTD is prevented. C, D, The experiment presented in B, was repeated using anisomycin (25 μm; n = 8) and emetine (20 μm; n = 8); protein synthesis inhibition prevents synaptic tagging in RLTD. Analog traces always represent typical field EPSPs 30 min before (dotted line) and 30 min (dashed line) and 6 h (solid line) after WLFS of input S1 or in cases in which WLFS was delivered to S2. The dashed arrows indicate the time point of the WLFS of the corresponding synaptic input.

Figure 3.

MAPK pathway during RLTD. A, Effect of Ras inhibition by manumycin-A on RLTD (n = 7). After recording a baseline for 50 min, manumycin-A (2 μm) was applied for 10 min before being coapplied with rolipram for another 1 h. WLFS in S1 in the presence of manumycin-A had no effect on RLTD (filled circles); control potentials from S2 remained stable (open circles). B, Effect of LT-82 (n = 7) on RLTD. This is the same experiment as in Figure 2C but with the presence of a Rap inhibitor, LT-82 (200 ng/ml), which prevents the RLTD (filled circles). C, Influence of the MEK inhibitor U0126 on RLTD (n = 7). After recording a baseline for 50 min, U0126 (1 μm) was applied for 10 min before being coapplied with rolipram for another 1 h. WLFS in S1 in the presence of MEK inhibitor U0126 prevents RLTD (filled circles); control recording of S2 (open circles) remained stable during the recorded period. D, PD98059 (1 μm; n = 7) confirmed the results obtained in C. Analog traces always represent typical field EPSPs 30 min before (dotted line) and 30 min (dashed line) and 6 h (solid line) after WLFS of input S1 or in cases in which WLFS was delivered to S2. Dashed arrows indicate the time point of the WLFS of the corresponding synaptic input.