Joanna M. Williams

Correlations between immediate early gene induction and the persistence of long-term potentiation.

The duration of long-term potentiation in the dentate gyrus of awake rats was examined following systematic manipulation of the number of stimulus trains delivered. This was correlated with the induction of immediate early genes in separate groups of animals given identical stimulus regimes. Following 10 trains of stimulation, long-term potentiation decayed with a time constant of up to several days (long-term potentiation 2), and this correlated with the appearance of an increase in the messenger RNA and protein levels of zif/268. Increasing the number of stimulus trains resulted in a greater probability of eliciting long-term potentiation with a time constant of several weeks (long-term potentiation 3), as well as increasing the induction of zif/268, c-Jun, Jun-B, Jun-D and Fos-related proteins. When 10 trains were delivered repeatedly on up to five consecutive days, only the zif/268 protein levels showed associated changes. These data provide support for the hypothesis that long-term potentiation 3 involves mechanisms additional to those for long-term potentiation 2. One possible mechanism is altered gene expression, initiated by immediate early gene transcription factors such as zif/268 and possibly homo- or heterodimers of Fos and Jun family members, that then contributes to the stabilization or maintenance of long-term potentiation 3.

LTP maintenance and its protein synthesis-dependence

The properties of long-term potentiation (LTP) mirror those of associative memory in a number of interesting ways. Although plasticity at monosynaptic connections is not expected to account for the varied subtle characteristics of distributed memories, nonetheless it is important to establish how far the parallels can be drawn. Here, we briefly address whether properties of LTP such as its duration, reversibility, savings and reconsolidation relate to corresponding memory phenomena. We then address whether LTP stabilization in fact requires protein synthesis, as this has been challenged in recent times much like the necessity for protein synthesis in the consolidation of long-term memory has been queried. We conclude that the case is still very strong for a necessary role of protein synthesis in LTP stabilization, even though the identities of the synthesized proteins and their contributions to the LTP process are not fully understood. However, we highlight areas of research that could be usefully conducted to further our understanding of the properties and protein synthesis-dependence of LTP.

Properties and Mechanisms of LTP Maintenance

Memory is fundamentally important to everyday life, and memory loss has devastating consequences to individuals and society. Understanding the neurophysiological and cellular basis of memory paves the way for gaining insights into the molecular steps involved in memory formation, thereby revealing potential therapeutic targets for neurological diseases. For three decades, long-term potentiation (LTP) has been the gold standard synaptic model for mammalian memory mechanisms, in large part because of its long-lasting nature. Here, the authors summarize the characteristics of LTP persistence in the dentate gyrus of the hippocampus, comparing this with other hippocampal subregions and neocortex. They consider how long LTP can last and how its persistence is affected by subsequent behavioral experiences. Next, they review the molecular mechanisms known to contribute to LTP induction and persistence, in particular the role of new gene expression and protein synthesis and how they may be associated with potential structural reorganization of the synapse. A temporal schema for the processes important for consolidating LTP into a persistent form is presented. The parallels between the molecular aspects of LTP and memory strongly support the continuation with LTP as a model system for studying the mechanisms underlying long-term memory consolidation and retention.

Krox20 may play a key role in the stabilization of long-term potentiation

Long-term potentiation-inducing stimulation of the perforant path was followed in dentate gyrus granule cells by a dramatic increase of mRNA and protein for Krox20, a zinc-finger-containing transcription factor. Induction of Krox20 required stimulation sufficient to induce LTP and was prevented by NMDA antagonists CPP and MK-801, which block LTP induction. Krox20 protein increased within 20 min of tetanization, was maximal between 1 and 8 h, and was still significantly elevated at 24 h after LTP induction. This prolonged appearance is in striking contrast with the more transient induction of the related molecule, Krox24. The elevation in the mRNA for Krox20 and Krox24 was of similar duration, suggesting that the Krox20 protein has a greater stability and may play a key role in the stabilization of long-term potentiation.

Calcium/Calmodulin-Dependent Protein Kinase II Mediates Group I Metabotropic Glutamate Receptor-Dependent Protein Synthesis and Long-Term Depression in Rat Hippocampus

Activation of Group I metabotropic glutamate receptors (mGluRs) in rat hippocampus induces a form of long-term depression (LTD) that is dependent on protein synthesis. However, the intracellular mechanisms leading to the initiation of protein synthesis and expression of LTD after mGluR activation are only partially understood. We investigated the role of several pathways linked to mGluR activation, translation initiation, and induction of LTD. We found that Group I mGluR-dependent protein synthesis and associated LTD, as induced by the agonist (RS)-3,5-dihydrophenylglycine (DHPG) or paired-pulse synaptic stimulation, was dependent on activation of calcium/calmodulin-dependent protein kinase IIα (CaMKII). DHPG induced a transient increase in the level of phospho-CaMKII (phospho-CaMKIIT286) in synaptoneurosomes prepared from whole hippocampus and in CA1 minislices. In synaptoneurosomes, DHPG also induced an increase in phosphorylation of eIF4E, and an increase in protein synthesis that was abolished by translation inhibitors and the CaMKII inhibitors 1-[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN62) and 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)amino-N-(4-chloro-cinnamyl)-N-methylbenzylamine (KN93). In field recordings from CA1, both the translation inhibitor cycloheximide and KN62 significantly reduced DHPG-induced LTD. Combined application did not further reduce the LTD, suggesting a common mechanism. In whole-cell recordings, a third CaMKII inhibitor, AIP (autocamtide-2-related inhibitory peptide), significantly reduced the DHPG-induced LTD of synaptic currents. Inhibition of the classical pathway mediating many Group I mGluR effects by blocking PKC (protein kinase C) or PLC (phospholipase C) did not impair DHPG-induced protein synthesis or LTD. Collectively, these findings demonstrate an important role for CaMKII in mediating the initiation of protein synthesis that then supports the postsynaptic expression of DHPG-induced LTD.

Long-term regulation of N-methyl-D-aspartate receptor subunits and associated synaptic proteins following hippocampal synaptic plasticity.

Synaptic plasticity in the dentate gyrus is dependent on activation of the N-methyl-D-aspartate (NMDA)-subtype of glutamate receptors. In this study, we show that synaptic plasticity in turn regulates NMDA receptors, since subunits of the NMDA receptor complex are bidirectionally and independently regulated in the dentate gyrus following activation of perforant synapses in awake animals. Low-frequency stimulation that produced a mild synaptic depression resulted in a decrease in the NMDA receptor subunits NR1 and NR2B 48 h following stimulation. High-frequency stimulation that produced long-term potentiation resulted in an increase in NR1 and NR2B at the same time point. Further investigations revealed that in contrast to NR2B, NR1 levels increased gradually after long-term potentiation induction, reaching a peak level at 48 h, and were insensitive to the competitive NMDA receptor antagonist 3-3(2-carboxypiperazin-4-yl) propyl-1-phosphate. The increased levels of NR1 and NR2B at 48 h were found associated with synaptic membranes and with increased NMDA receptor-associated proteins, postsynaptic density protein 95, neuronal nitric oxide synthase and Ca(2+)/calmodulin-dependent protein kinase II, alpha subunit. These data suggest that the persistence of long-term potentiation is associated with an increase in the number of NMDA receptor complexes, which may be indicative of an increase in synaptic contact area.

Biphasic changes in the levels of N-methyl-d-aspartate receptor-2 subunits correlate with the induction and persistence of long-term potentiation

N-Methyl-D-aspartate glutamate receptors (NMDAR) form ion channels made up of polypeptides from two classes of subunits; NR1 is obligatory for function whereas members of the NR2 class regulate the properties of the channel. Long-term potentiation (LTP) of synaptic transmission is an event largely dependent on NMDAR activation, and is studied as the primary cellular model of memory in the mammalian brain. While there has been a focus on non-NMDARs in mediating the expression of LTP, we report here biochemical evidence for plasticity of the NMDAR that is associated with LTP persistence in awake animals. Following the establishment of LTP in perforant path synapses of the dentate gyrus, we observed a rise in NR2B protein levels 48 h post-tetanus which was dependent upon activation of NMDARs during the tetanization, and which strongly correlated with the degree of LTP measured at this time-point. We also observed a transient increase in both NR2B and NR2A protein levels 20 min post-tetanus that returned to control levels by 4 h. These early increases were not observed in anaesthetized animals which do not sustain persistent LTP. Our data demonstrate a marked plasticity of NMDAR subunit expression, which may affect LTP persistence, as well as the subsequent ability to induce LTP at previously activated synapses.

Differential Trafficking of AMPA and NMDA Receptors during Long-Term Potentiation in Awake Adult Animals

Despite a wealth of evidence in vitro that AMPA receptors are inserted into the postsynaptic membrane during long-term potentiation (LTP), it remains unclear whether this occurs in vivo at physiological concentrations of receptors. To address the issue of whether native AMPA or NMDA receptors undergo such trafficking during LTP in the adult brain, we examined the synaptic and surface expression of glutamate receptor subunits during the early induction phase of LTP in the dentate gyrus of awake adult rats. Induction of LTP was accompanied by a rapid NMDA receptor-dependent increase in surface expression of glutamate receptor 1–3 (GluR1–3) subunits. However, in the postsynaptic density fraction only GluR1 accumulated. GluR2/3-containing AMPA receptors, in contrast, were targeted exclusively to extrasynaptic sites in a protein synthesis-dependent manner. NMDA receptor subunits exhibited a delayed accumulation, both at the membrane surface and in postsynaptic densities, that was dependent on protein synthesis. These data suggest that trafficking of native GluR1-containing AMPA receptors to synapses is important for early-phase LTP in awake adult animals, and that this increase is followed homeostatically by a protein synthesis-dependent trafficking of NMDA receptors.

Synaptic activity-dependent modulation of mitochondrial gene expression in the rat hippocampus.

In order to identify genes that may underlie the maintenance of long-term potentiation (LTP) at perforant path synapses, complementary DNA libraries were synthesised from dentate gyrus total RNA extracts prepared 48 h after the induction of LTP and from control dentate gyrus extracts. Through differential screening of the LTP library we have identified the mitochondrial 12S rRNA (mt12SrRNA) as a transcript that was elevated at this late time. Northern blot analyses showed that the elevation in mt12SrRNA expression began around 8 h and persisted for at least 2 weeks post-tetanus. We then examined the expression patterns of other mitochondrially-encoded genes and demonstrated a similar elevation in their expression. mt12SrRNA levels were also elevated in other hippocampal regions, including areas CA3 and CA1 and were elevated following low-frequency stimulation or in the presence of an N-methyl-D-aspartate receptor antagonist where induction of LTP was precluded. Taken together, these observations suggest that a long-lasting up-regulation of energy production may be triggered by synaptic activity and this activity need not be of sufficient strength to induce LTP, but may be related to the induction of a metaplastic state.

Temporal Profiling of Gene Networks Associated with the Late Phase of Long-Term Potentiation In Vivo

Long-term potentiation (LTP) is widely accepted as a cellular mechanism underlying memory processes. It is well established that LTP persistence is strongly dependent on activation of constitutive and inducible transcription factors, but there is limited information regarding the downstream gene networks and controlling elements that coalesce to stabilise LTP. To identify these gene networks, we used Affymetrix RAT230.2 microarrays to detect genes regulated 5 h and 24 h (n = 5) after LTP induction at perforant path synapses in the dentate gyrus of awake adult rats. The functional relationships of the differentially expressed genes were examined using DAVID and Ingenuity Pathway Analysis, and compared with our previous data derived 20 min post-LTP induction in vivo. This analysis showed that LTP-related genes are predominantly upregulated at 5 h but that there is pronounced downregulation of gene expression at 24 h after LTP induction. Analysis of the structure of the networks and canonical pathways predicted a regulation of calcium dynamics via G-protein coupled receptors, dendritogenesis and neurogenesis at the 5 h time-point. By 24 h neurotrophin-NFKB driven pathways of neuronal growth were identified. The temporal shift in gene expression appears to be mediated by regulation of protein synthesis, ubiquitination and time-dependent regulation of specific microRNA and histone deacetylase expression. Together this programme of genomic responses, marked by both homeostatic and growth pathways, is likely to be critical for the consolidation of LTP in vivo.