Julietta U. Frey

Reward-related fMRI activation of dopaminergic midbrain Is associated with enhanced hippocampus- dependent long-term memory formation

Long-term potentiation in the hippocampus can be enhanced and prolonged by dopaminergic inputs from midbrain structures such as the substantia nigra. This improved synaptic plasticity is hypothesized to be associated with better memory consolidation in the hippocampus. We used a condition that reliably elicits a dopaminergic response, reward anticipation, to study the relationship between activity of dopaminergic midbrain areas and hippocampal long-term memory in healthy adults. Pictures of object drawings that predicted monetary reward were associated with stronger fMRI activity in reward-related brain areas, including the substantia nigra, compared with non-reward-predicting pictures. Three weeks later, recollection and source memory were better for reward-predicting than for non-reward-predicting pictures. FMRI activity in the hippocampus and the midbrain was higher for reward-predicting pictures that were later recognized compared with later forgotten pictures. These data are consistent with the hypothesis that activation of dopaminergic midbrain regions enhances hippocampus-dependent memory formation, possibly by enhancing consolidation.

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.

Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon.

Mutant mice lacking the central region of the presynaptic active zone protein Bassoon were generated to establish the role of this protein in the assembly and function of active zones as sites of synaptic vesicle docking and fusion. Our data show that the loss of Bassoon causes a reduction in normal synaptic transmission, which can be attributed to the inactivation of a significant fraction of glutamatergic synapses. At these synapses, vesicles are clustered and docked in normal numbers but are unable to fuse. Phenotypically, the loss of Bassoon causes spontaneous epileptic seizures. These data show that Bassoon is not essential for synapse formation but plays an essential role in the regulated neurotransmitter release from a subset of glutamatergic synapses.

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.

Requirement of β-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 β‐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.

Plasticity-specific phosphorylation of CaMKII, MAP-kinases and CREB during late-LTP in rat hippocampal slices in vitro

Processes of learning, memory formation and functional plasticity, such as long-term potentiation (LTP) are associated with activity changes of alphaCaMKII, MAPKs and CREB proteins. Little is known on the temporal regulation of the phosphorylation states of these proteins during late-LTP, a period which requires the synthesis of new macromolecules and has been postulated to correlate with the consolidation of a memory trace at the cellular level. We now present data from hippocampal slices in vitro obtained from adult rats that describe such specific phosphorylation changes of the above mentioned three molecules during early- and late-LTP. We detail that LTP induction and its maintenance reveals a delayed onset of continuous CREB phosphorylation with two separate peaks at 45 min and 6 h before it decays back to baseline phosphorylation levels; whereas alphaCaMKII and MAPK2 remain in a specific, but enhanced phosphorylation state throughout the induction, early- and late-LTP. These LTP-regulated phosphorylation events were NMDAR-dependent and upon the activity of a translated serine-threonine kinase. Interestingly, only the late enhancement of pCREB was clearly dependent on protein synthesis. Our data extend results describing the regulation of alphaCaMKII, MAPKs and CREB phosphorylation during early stages of LTP, suggesting a specific role of these enzymes also during phases of LTP consolidation in adult animals.

Long-Term Effects of Brief Acute Stress on Cellular Signaling and Hippocampal LTP

In a previous study, we reported that a brief exposure to swim stress transforms an electrically induced, protein synthesis-independent early long-term potentiation (early LTP) into a protein synthesis-dependent late LTP [“reinforcement of LTP” in the hippocampal dentate gyrus (DG)] (Korz and Frey, 2003). This transformation depends on activation of mineralocorticoid receptors (MRs) by corticosterone, and on intact basolateral amygdala (BLA) function. Here, we demonstrate that a brief swim experience results in lasting changes in levels of hippocampal cellular signaling molecules that are known to be involved in the induction of late LTP. Within the DG, MRs were rapidly upregulated, whereas glucocorticoid receptor (GR) levels were elevated with a 3 h delay. Levels of phosphorylated mitogen-activated protein kinase 2 (pMAPK2) and p38 MAPK, as well as phosphorylated calcium/calmodulin-dependent protein kinase II (pCaMKII) were enhanced shortly after swim stress and remained elevated until 24 h, whereas levels of phosphorylated cAMP response element-binding protein (pCREB) remained unchanged. MR and GR were upregulated with a longer delay within the CA1 region, whereas levels of pMAPK2 and p38MAPK were rapidly increased, but the former returned to basal levels after 3 h. Levels of pCREB and pCaMKII were maintained in an enhanced state after swim stress. DG-LTP reinforcement requires a serotonergic but not dopaminergic heterosynaptic receptor activation that probably mediates the BLA-dependent modulation of LTP under stress. Thus, molecular alterations induced by specific stress resemble late LTP-related molecular changes. These changes, in interaction with stress-specific heterosynaptic processes, may support the transformation of early LTP into late LTP. The results contribute to the understanding of the rapid consolidation of cellular and possibly systemic memories triggered by stress.

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.