PKM zeta maintains late long-term potentiation by N-ethylmaleimide-sensitive factor/GluR2-dependent trafficking of postsynaptic AMPA receptors.

Although the maintenance mechanism of late long-term potentiation (LTP) is critical for the storage of long-term memory, the expression mechanism of synaptic enhancement during late-LTP is unknown. The autonomously active protein kinase C isoform, protein kinase Mzeta (PKMzeta), is a core molecule maintaining late-LTP. Here we show that PKMzeta maintains late-LTP through persistent N-ethylmaleimide-sensitive factor (NSF)/glutamate receptor subunit 2 (GluR2)-dependent trafficking of AMPA receptors (AMPARs) to the synapse. Intracellular perfusion of PKMzeta into CA1 pyramidal cells causes potentiation of postsynaptic AMPAR responses; this synaptic enhancement is mediated through NSF/GluR2 interactions but not vesicle-associated membrane protein-dependent exocytosis. PKMzeta may act through NSF to release GluR2-containing receptors from a reserve pool held at extrasynaptic sites by protein interacting with C-kinase 1 (PICK1), because disrupting GluR2/PICK1 interactions mimic and occlude PKMzeta-mediated AMPAR potentiation. During LTP maintenance, PKMzeta directs AMPAR trafficking, as measured by NSF/GluR2-dependent increases of GluR2/3-containing receptors in synaptosomal fractions from tetanized slices. Blocking this trafficking mechanism reverses established late-LTP and persistent potentiation at synapses that have undergone synaptic tagging and capture. Thus, PKMzeta maintains late-LTP by persistently modifying NSF/GluR2-dependent AMPAR trafficking to favor receptor insertion into postsynaptic sites.

Synergistic requirements for the induction of dopaminergic D1/D5-receptor-mediated LTP in hippocampal slices of rat CA1 in vitro.

Dopaminergic D1/D5-receptor-mediated processes are important for certain forms of memory and its cellular model, i.e. hippocampal long-term potentiation (LTP) in CA1. D1/D5-receptor function is required for the induction of the protein synthesis-dependent maintenance of CA1-LTP (late-LTP) by activating the cAMP/PKA-pathway. In earlier studies we had reported a synergistic interaction of D1/D5-receptor function and N-methyl-D-aspartate (NMDA)-receptors (Frey, 2001, Long-lasting hippocampal plasticity: cellular model for memory consolidation? In: Richter, D. (Ed.), Cell Polarity and Subcellular RNA Localization. Springer-Verlag, Berlin-Heidelberg, pp. 27-40). Interestingly, the short-term application of D1/D5-receptor agonists (SKF38393 or 6-bromo-APB, 50 microM) can induce a slow-onset potentiation. This D1/D5-agonist-induced delayed-onset potentiation (D1/D5-LTP) resembles late-LTP, i.e. it is dependent on protein synthesis in the CA1 of rat hippocampal slices in vitro. The question arises as to whether D1/D5-LTP also requires glutamatergic stimulation, i.e. NMDA-receptor activation. We provide first evidence that a synergistic role of D1/D5- as well as NMDA-receptor-function is required in mediating processes relevant for the maintenance of this protein synthesis-dependent potentiation.

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.

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).

Protein synthesis-dependent long-term functional plasticity: methods and techniques.

There is growing interest in late-LTP and late-LTD, that is, distinct forms of functional plasticity that require somatic functions such as protein synthesis in addition to the transient synaptic processes that are required for short lasting forms. Interestingly, to date only these forms of lasting plastic events could be detected in healthy, freely moving animals and thus, they are considered as physiological cellular models of learning and memory formation. Late-LTP and -LTD are characterized by 'synaptic tagging' or 'capture' and 'synaptic cross-tagging', but there are only a few laboratories that can currently perform experiments studying these properties. In brain slice work, there are many different approaches to investigate these processes using different methodological variations: some allow slices to rest for long periods before the experiment starts, others do not; some run their experiments at near to physiological temperatures, others at lower temperatures; some stimulate frequently, others do not.

Synaptic tagging and cross-tagging: the role of protein kinase Mzeta in maintaining long-term potentiation but not long-term depression.

Protein kinase Mzeta (PKMzeta) is a persistently active protein kinase C isoform that is synthesized during long-term potentiation (LTP) and is critical for maintaining LTP. According to "synaptic tagging," newly synthesized, functionally important plasticity-related proteins (PRPs) may prolong potentiation not only at strongly tetanized pathways, but also at independent, weakly tetanized pathways if synaptic tags are set. We therefore investigated whether PKMzeta is involved in tagging and contributes to a sustained potentiation by providing strong and weak tetanization to two independent pathways and then disrupting the function of the kinase by a selective myristoylated zeta-pseudosubstrate inhibitory peptide. We found that persistent PKMzeta activity maintains potentiated responses, not only of the strongly tetanized pathway, but also of the weakly tetanized pathway. In contrast, an independent, nontetanized pathway was unaffected by the inhibitor, indicating that the function of PKMzeta was specific to the tagged synapses. To further delineate the specificity of the function of PKMzeta in synaptic tagging, we examined synaptic "cross-tagging," in which late LTP in one input can transform early into late long-term depression (LTD) in a separate input or, alternatively, late LTD in one input can transform early into late LTP in a second input, provided that the tags of the weak inputs are set. Although the PKMzeta inhibitor reversed late LTP, it did not prevent the persistent depression at the weakly stimulated, cross-tagged LTD input. Conversely, although the agent did not reverse late LTD, it blocked the persistent potentiation of weakly tetanized, cross-tagged synapses. Thus, PKMzeta is the first LTP-specific PRP and is critical for the transformation of early into late LTP during both synaptic tagging and cross-tagging.

The type IV-specific phosphodiesterase inhibitor rolipram and its effect on hippocampal long-term potentiation and synaptic tagging.

We investigated the effects of rolipram, a selective cAMP phosphodiesterase (PDE) inhibitor, on late plastic events during functional CA1 plasticity in vitro in rat hippocampal slices. We present data showing that an early form of long-term potentiation (LTP) (early-LTP) that normally decays within 2-3 hr can be converted to a lasting LTP (late-LTP) if rolipram is applied during tetanization. This rolipram-reinforced LTP (RLTP) was NMDA receptor and protein synthesis dependent. cAMP formation in region CA1 during late-LTP requires dopaminergic receptor activity (Frey et al., 1989, 1990). Thus, we studied whether RLTP was influenced by inhibitors of the D(1)/D(5) receptor. Application of the specific D(1)/D(5) antagonist SCH23390 (0.1 microm) did not prevent RLTP, suggesting that the phosphodiesterase inhibitor acts downstream of the D(1)/D(5) receptors. We also studied whether rolipram can interact with processes of synaptic tagging, because RLTP was also dependent on protein synthesis, similar to late-LTP. Inhibition of PDE and subsequent induction of RLTP in one synaptic population were able to transform early-LTP into late-LTP in a second, independent synaptic population of the same neurons. This supports our hypothesis that cAMP-dependent processes are directly involved in the synthesis of plasticity-related proteins.

Requirement of beta-adrenergic receptor activation and protein synthesis for LTP-reinforcement by novelty in rat dentate gyrus.

Long-term potentiation (LTP) is supposed to be a cellular mechanism involved in memory formation. Similar to distinct types of memory formation, LTP can be separated into a protein synthesis-independent early phase (early-LTP) and a protein synthesis-dependent late phase (late-LTP). An important question is whether the transformation from early- into late-LTP can be elicited by behavioural conditions such as the attention to novel events. Therefore, we investigated the effect of exploration of a novel environment (novelty-exploration) on subsequently induced early-LTP in the dentate gyrus of freely moving rats. While a delay of 60 min between exploration onset and LTP induction had no effect, intervals of 30 or 15 min led to a reinforcement of early- to late-LTP. Exploration of a familiar environment failed to prolong LTP maintenance. The novelty-induced LTP reinforcement was blocked when the translation inhibitor anisomycin or the beta-adrenergic antagonist propranolol were applied intracerebroventricularly before exploration onset. These findings support the hypothesis that the synergistic interplay of novelty-triggered noradrenergic activity and weak tetanic stimulation promotes the synthesis of certain proteins that are required for late-LTP. Such a cellular mechanism may underlie novelty-dependent enhancement of memory formation.

Time-dependent depotentiation in the dentate gyrus of freely moving rats by repeated brief 7 Hz stimulation.

Long-term potentiation (LTP) in the hippocampus of awake animals is known to be reversed by long trains of low-frequency stimulation (LFS). Here we show that a persistent LTP reversal, i.e. depotentiation (DP), can be induced in the dentate gyrus of freely moving rats by brief 7 Hz stimulations (three trains of 100 pulses, 1 min intertrain-interval) if LFS was applied 2 min after tetanization. In contrast, the same LFS protocol given 15 min after tetanization failed to induce long-lasting DP. Likewise, reduction of LFS (given 2 min post-tetanus) to two trains resulted only in short-term DP. Because naturally occurring hippocampal 7 Hz oscillatory events are correlated to new sensory input, our results might be relevant for the understanding of the mechanisms of LTP reversal induced by processing new information.