Sreedharan Sajikumar

PKMζ 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 Mζ (PKMζ), is a core molecule maintaining late-LTP. Here we show that PKMζ 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 PKMζ 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. PKMζ 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 PKMζ-mediated AMPAR potentiation. During LTP maintenance, PKMζ 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, PKMζ maintains late-LTP by persistently modifying NSF/GluR2-dependent AMPAR trafficking to favor receptor insertion into postsynaptic sites.

Synaptic Tagging and Cross-Tagging: The Role of Protein Kinase Mζ in Maintaining Long-Term Potentiation But Not Long-Term Depression

Protein kinase Mζ (PKMζ) 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 PKMζ 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 ζ-pseudosubstrate inhibitory peptide. We found that persistent PKMζ 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 PKMζ was specific to the tagged synapses. To further delineate the specificity of the function of PKMζ 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 PKMζ 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, PKMζ is the first LTP-specific PRP and is critical for the transformation of early into late LTP during both synaptic tagging and cross-tagging.

Identification of Compartment- and Process-Specific Molecules Required for “Synaptic Tagging” during Long-Term Potentiation and Long-Term Depression in Hippocampal CA1

Protein synthesis-dependent forms of hippocampal long-term potentiation (late LTP) and long-term depression (late LTD) are prominent cellular mechanisms underlying memory formation. Recent data support the hypothesis that neurons store relevant information in dendritic functional compartments during late LTP and late LTD rather than in single synapses. It has been suggested that processes of “synaptic tagging” are restricted to such functional compartments. Here, we show that in addition to apical CA1 dendrites, synaptic tagging also takes place within basal CA1 dendritic compartments after LTP induction. We present data that tagging in the basal dendrites is restricted to these compartments. Plasticity-related proteins, partially nonspecific to the locally induced process, are synthesized in dendritic compartments and then captured by local, process-specific synaptic tags. We support these findings in two ways: (1) late LTP/LTD, locally induced in apical or basal (late LTP) dendrites of hippocampal CA1 neurons, does not spread to the basal or apical compartment, respectively; (2) the specificity of the synaptic plasticity event is achieved by the activation of process- and compartment-specific synaptic tag molecules. We have identified calcium/calmodulin-dependent protein kinase II as the first LTP-specific and extracellular signal-regulated kinase 1/2 as LTD-specific tag molecules in apical dendritic CA1 compartments, whereas either protein kinase A or protein kinase Mζ mediates LTP-specific tags in basal dendrites.

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.

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 D1/D5 receptor. Application of the specific D1/D5 antagonist SCH23390 (0.1 μm) did not prevent RLTP, suggesting that the phosphodiesterase inhibitor acts downstream of the D1/D5 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.

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.

Metaplasticity governs compartmentalization of synaptic tagging and capture through brain-derived neurotrophic factor (BDNF) and protein kinase Mζ (PKMζ)

Activity-dependent synaptic plasticity is widely accepted to be the cellular correlate of learning and memory. It is believed that associativity between different synaptic inputs can transform short-lasting forms of synaptic plasticity (<3 h) to long-lasting ones. Synaptic tagging and capture (STC) might be able to explain this heterosynaptic support, because it distinguishes between local mechanisms of synaptic tags and cell-wide mechanisms responsible for the synthesis of plasticity-related proteins (PRPs). STC initiate storage processes only when the strength of the synaptic tag and the local concentration of essential proteins are above a certain plasticity threshold. We present evidence that priming stimulation through the activation of metabotropic glutamate receptors substantially increases the “range of threshold” for functional plasticity by producing protein kinase Mζ (PKMζ) as a PRP through local protein synthesis. In addition, our results implicate BDNF as a PRP which is mandatory for establishing cross-capture between synaptic strengthening and weakening, whereas the newly generated PKMζ specifically establishes synaptic tagging of long-term potentiation. Most intriguingly, we show here that STC are confined to specific dendritic compartments and that these compartments contain “synaptic clusters” with different plasticity thresholds. Our results suggest that within a dendritic compartment itself a homeostatic process exists to adjust plasticity thresholds. The range in which these clusters operate can be altered by processes of metaplasticity, which will operate on the cluster independently of other clusters at the same dendrite. These clusters will then prepare the synaptic network to form long-term memories.

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 D1 and D5 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 D1 and D5 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).

Dopamine Induces LTP Differentially in Apical and Basal Dendrites through BDNF and Voltage-Dependent Calcium Channels.

The dopaminergic modulation of long-term potentiation (LTP) has been studied well, but the mechanism by which dopamine induces LTP (DA-LTP) in CA1 pyramidal neurons is unknown. Here, we report that DA-LTP in basal dendrites is dependent while in apical dendrites it is independent of activation of L-type voltage-gated calcium channels (VDCC). Activation via NMDAR is critical for the induction of DA-LTP in both apical and basal dendrites, but only BDNF is required for the induction and maintenance of DA-LTP in apical dendrites. We report that dopaminergic modulation of LTP is lamina-specific at the Schaffer collateral/commissural synapses in the CA1 region.

Protein kinase Mζ is essential for the induction and maintenance of dopamine-induced long-term potentiation in apical CA1 dendrites

Dopaminergic D1/D5-receptor-mediated processes are important for certain forms of memory as well as for a cellular model of memory, hippocampal long-term potentiation (LTP) in the CA1 region of the hippocampus. D1/D5-receptor function is required for the induction of the protein synthesis-dependent maintenance of CA1-LTP (L-LTP) through activation of 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 for L-LTP. Furthermore, we have found the requirement of the atypical protein kinase C isoform, protein kinase Mζ (PKMζ) for conventional electrically induced L-LTP, in which PKMζ has been identified as a LTP-specific plasticity-related protein (PRP) in apical CA1-dendrites. Here, we investigated whether the dopaminergic pathway activates PKMζ. We found that application of dopamine (DA) evokes a protein synthesis-dependent LTP that requires synergistic NMDA-receptor activation and protein synthesis in apical CA1-dendrites. We identified PKMζ as a DA-induced PRP, which exerted its action at activated synaptic inputs by processes of synaptic tagging.