MAPK establishes a molecular context that defines effective training patterns for long-term memory formation

Abstract

Although the importance of spaced training trials in the formation of long-term memory (LTM) is widely appreciated, surprisingly little is known about the molecular mechanisms that support interactions between individual trials. The intertrial dynamics of ERK/MAPK activation have recently been correlated with effective training patterns for LTM. However, whether and how MAPK is required to mediate intertrial interactions remains unknown. Using a novel two-trial training pattern which induces LTM in Aplysia, we show that the first of two training trials recruits delayed protein synthesis-dependent nuclear MAPK activity that establishes a unique molecular context involving the recruitment of CREB kinase and ApC/EBP and is an essential intertrial signaling mechanism for LTM induction. These findings provide the first demonstration of a requirement for MAPK in the intertrial interactions during memory formation and suggest that the kinetics of MAPK activation following individual experiences determines effective training intervals for LTM formation.

Figure 1.

Long-term memory for sensitization of the tail-elicited tail withdrawal reflex has a narrow temporal two-trial training window. a, Schematic of the reduced behaving preparation used to study the tail-elicited tail withdrawal reflex (inset: representative tail withdrawal [TW] response to test stimulation [TS] as measured by a strain gauge). b, Summary of average 18–22 h (LTM) scores in preparations trained with two shocks spaced by 15 min (2×15), 45 min (2×45), 60 min (2×60), or untrained (NS). Data are presented as mean ± SEM. Asterisk indicates enhanced responding at 18–22 h in 2×45 trained animals over untrained controls (unpaired t test).

Figure 2.

Serotonin can serve as a proxy for training shock in the restricted temporal activation of protein synthesis-dependent MAPK. a, A 5 min 5HT exposure to the isolated pleural-pedal ganglia induced significant MAPK activation in tail SNs at 45 min compared with paired ASW-treated control ganglia. No activation was observed at 15 min or at 1 h following the same treatment. b, Exposure to the protein synthesis inhibitor emetine (eme.) for 30 min before 5HT treatment prevented the 5HT-induced MAPK activation at 45 min.

Figure 3.

Trial 1 MAPK translocates to the nucleus of tail sensory neurons. ASW- and 5HT-treated pleural ganglia were fixed beginning 45 min after the onset of treatment and labeled for phosphorylated MAPK (P-MAPK). DAPI labeling identifies cell nuclei. Left, Representative confocal microscopy images through the VC cluster of sensory neurons. Arrowheads identify cells with representative nuclear translocation. Scale bar, 50 μm. Right, Summary analysis of nuclear/cytoplasmic ratio of P-MAPK immunofluorescence. Asterisk indicates a significant increase in the nuclear/cytoplasmic ratio at 45 min post-5HT. Number of Vc clusters/total number of cells analyzed are indicated.

Figure 4.

Trial 1 MAPK activates the CREB kinase p90rsk and increases transcription of the immediate early gene ApC/EBP. a, Top, Representative image of phosphorylated p90rsk (p-p90rsk) levels in ASW- and 5HT-treated SN clusters at 45 min. Bottom, Summary analysis of normalized p-p90rsk levels observed in ASW- and 5HT-treated SNs. Exposure to the MEK inhibitor, U0126, disrupted the observed 5HT-induced increase in p-p90rsk. Treatment with the inactive analog, U0124, had no significant effect. Data in histograms are presented as mean ± SEM. b, qRT-PCR was performed to determine transcript levels of the immediate early gene ApC/EBP and the ribosomal S4 subunit. Levels of ApC/EBP (normalized to levels of S4) were increased by 5HT. U0126, but not U0124, disrupted the 5HT-induced ApC/EBP induction. Asterisks indicate significant within-group activation. Data in histograms are presented as median ± interquartile range.

Figure 5.

Trial 1 MAPK is necessary for the intertrial interaction during induction of LTM. a, Experimental strategy to selectively disrupt MAPK activation during the initial training shock (ASW histogram), while leaving intact the potential for MAPK activation by the second shock during two-trial training. I, CNS exposure to U0126 for 40 min before, during and 15 min after the administration of a single training shock to behaving T-TWR preparations was sufficient to disrupt Trial 1 MAPK activation in tail SNs (ASW vs I in summary histogram, right). II, A 30 min drug washout following the same treatment in I (55 min U0126 exposure, no training), was sufficient to restore MEK activity as demonstrated by the ability of a single shock at this time point to activate MAPK at 45 min postshock. b, The specific disruption of Trial 1-induced MAPK activation during two-trial learning prevents LTM formation. Asterisk indicates a significant difference in the between group comparison. Data are mean ± SEM.

Figure 6.

A model depicting how MAPK establishes a molecular context that defines effective training intervals for LTM. A single training shock (TS) releases 5HT onto tail SNs and sensorimotor synapses (Marinesco and Carew, 2002; Philips et al., 2011). 5HT acts at SN membranes through a receptor-coupled adenylate cyclase to increase levels of cAMP and activate PKA in sensory neuron somata and synaptic terminals (Bacskai et al., 1993; Muller and Carew, 1998). cAMP/PKA activation is transient and is inactivated within 15 min following TS. MAPK activation by the same TS is delayed, and appears in the cell body and nucleus at 45 min, but not at 15 min or 1 h. Trial 1 MAPK activation requires protein synthesis which may account, at least in part, for its delayed activation. Trial 1 MAPK activity establishes a unique molecular context within tail SNs by 45 min that is required for two-trial LTM formation. By 45 min, MAPK has activated the CREB pathway as observed in the increased phosphorylation of the CREB kinase p90rsk and increased ApC/EBP expression. By 1 h, the MAPK-dependent molecular context has ended. Although required for LTM, this MAPK-dependent molecular context is not sufficient for LTM and must interact with additional signaling from a second TS at 45 min to induce LTM (two-trial LTM induction). The necessary signaling provided by the second TS during two-trial LTM induction may be the recruitment of additional 5HT release, PKA activity and the PKA-dependent activation of the ApC/EBP/APAF complex which is sufficient to drive long-term synaptic facilitation at SN–MN synapses (Lee et al., 2006). The delayed onset of the MAPK-dependent molecular context may be sufficient to explain the failure of the short (15 min) training interval to support LTM formation. Longer (1 h) training intervals may similarly fail to support LTM induction because the required MAPK signaling and MAPK-dependent gene expression has ended by 1 h.