Bilateral histone deacetylase 1 and 2 activity and enrichment at unique genes following induction of long-term potentiation in vivo.

Long-term potentiation (LTP) is a synaptic plasticity mechanism critical to long-term memory. LTP induced in vivo is characterized by altered transcriptional activity, including a period of upregulation of gene expression which is followed by a later dominant downregulation. This temporal shift to downregulated gene expression is predicted to be partly mediated by epigenetic inhibitors of gene expression, such as histone deacetylases (HDACs). Further, pharmacological inhibitors of HDAC activity have previously been shown to enhance LTP persistence in vitro. To explore the contribution of HDACs to the persistence of LTP in vivo, we examined HDAC1 and HDAC2 activity over a 24 hr period following unilateral LTP induction in the dentate gyrus of freely moving rats. Surprisingly, we found significant changes in HDAC1 and HDAC2 activity in both the stimulated as well as the unstimulated hemispheres, with the largest increase in activity occurring bilaterally, 20 min after LTP stimulation. During this time point of heightened activity, chromatin immunoprecipitation assays showed that both HDAC1 and HDAC2 were enriched at distinct sets of genes within each hemispheres. Further, the HDAC inhibitor Trichostatin A enhanced an intermediate phase of LTP lasting days, which has not previously been associated with altered transcription. The inhibitor had no effect on the persistence of LTP lasting weeks. Together, these data suggest that HDAC activity early after the induction of LTP may negatively regulate plasticity-related gene expression that is involved in the initial stabilization of LTP, but not its long-term maintenance.

Docosahexaenoic acid prevents palmitate-induced insulin-dependent impairments of neuronal health.

The importance of fatty acids (FAs) for healthy brain development and function has become more evident in the past decades. However, most studies focus on the hypothalamus as an important FA-sensing brain region involved in energy homeostasis. Less work has been done to evaluate the effects of FAs on brain regions such as the hippocampus or cortex, two important centres of learning, memory formation, and cognition. Furthermore, the mechanisms of how FAs modulate the neuronal development and function are incompletely understood. Therefore, this study examined the effects of the saturated FA palmitic acid (PA) and the polyunsaturated FA docosahexaenoic acid (DHA) on primary hippocampal and cortical cultures isolated from P0/P1 Sprague Dawley rat pups. Exposure to PA, but not DHA, resulted in severe morphological changes in primary neurons such as cell body swelling, axonal and dendritic blebbing, and a reduction in synaptic innervation, compromising healthy cell function and excitability. Pharmacological assessment revealed that the PA-mediated alterations were caused by overactivation of neuronal insulin signaling, demonstrated by insulin stimulation and phosphoinositide 3-kinase inhibition. Remarkably, co-exposure to DHA prevented all PA-induced morphological changes. This work provides new insights into how FAs can affect the cytoskeletal rearrangements and neuronal function via modulation of insulin signaling.

Glutamate Receptor Trafficking and Protein Synthesis Mediate the Facilitation of LTP by Secreted Amyloid Precursor Protein-Alpha.

Secreted amyloid precursor protein-alpha (sAPPα) has growth factor-like properties and can modulate long-term potentiation (LTP) and memory. Here, we demonstrate that exposure to sAPPα converts short-lasting LTP into protein-synthesis-dependent late LTP in hippocampal slices from male rats. sAPPß had no discernable effect. We hypothesized that sAPPα facilitated LTP via regulated glutamate receptor trafficking and de novo protein synthesis. We found using a linear mixed model that sAPPα stimulated trafficking of GluA2-lacking AMPARs, as well as NMDARs to the extrasynaptic cell surface, in a calcium/calmodulin-dependent kinase II and protein kinase G-dependent manner. Both cell surface receptor accumulation and LTP facilitation were present even after sAPPα washout and inhibition of receptor trafficking or protein synthesis prevented all these effects. Direct visualization of newly synthesized proteins (FUNCAT-PLA) confirmed the ability of sAPPα to stimulate de novo protein synthesis and revealed GluA1 as one of the upregulated proteins. Therefore, sAPPα generates a coordinated synthesis and trafficking of glutamate receptors to the cell surface that facilitate LTP.SIGNIFICANCE STATEMENT Secreted amyloid precursor protein-alpha (sAPPα) is a neurotrophic and neuroprotective protein that can promote synaptic plasticity and memory, yet the molecular mechanisms underlying these effects are still not well understood. Here, we show that sAPPα facilitates long-term potentiation (LTP) in a concentration-dependent fashion through cellular processes involving de novo protein synthesis and trafficking of both GluA2-lacking AMPARs and NMDARs to the extrasynaptic cell surface. sAPPα also enhances GluA1, but not GluA2, synthesis. The trafficking effects, along with the LTP facilitation, persist after sAPPα washout, revealing a metaplastic capability of exogenous sAPPα administration. sAPPα thus facilitates LTP through coordinated activation of protein synthesis and trafficking of glutamate receptors to the cell surface, where they are positioned for priming LTP.

Apoptotic cell death and temporal expression of apoptotic proteins Bcl-2 and Bax in the hippocampus, following binge ethanol in the neonatal rat model.

BACKGROUND: Binge-like ethanol (EtOH) exposure during the early rat neonatal period results in acute cell loss in specific brain regions, but such acute cell death has not been well established in the hippocampus. Binge alcohol exposure can also result in protein expression changes in the cerebellum that could alter cell fate, but this has not been reported for the hippocampal subregions. This study investigates acute apoptotic cell death in hippocampal regions CA1, CA3, and dentate gyrus (DG) following a binge EtOH exposure on postnatal day (PN) 6, PN8, or PN6 + 8 and the alteration in pro- and anti-apoptotic proteins following a single EtOH binge on PN6. METHODS: Apoptotic cell death was quantified 12 hours after EtOH binge exposure using the optical fractionator method. Western blot analysis determined expression of pro-apoptotic Bax and anti-apoptotic Bcl-2, 12, 24, and 48 hours after binge EtOH exposure on PN6. The Bcl-2:Bax ratio was used as a measure of vulnerability to apoptosis. RESULTS: Acute apoptosis increased significantly 12 hours following PN6 or 8 EtOH exposure in CA1, CA3, and DG, but the magnitude of apoptotic cell death was significantly greater in CA1 than in CA3 and DG, which did not differ. Significant cell death was not detected when a PN8 EtOH exposure was preceded by exposure on PN6. Binge EtOH exposure on PN6 resulted in a significant increase in expression of Bcl-2 and the Bcl-2:Bax ratio in the CA1/DG region at 24 hours after EtOH exposure on PN6. The Bcl-2:Bax ratio in the CA3 region was not altered. CONCLUSIONS: This study shows that repeated binge exposure does not have a cumulative effect on the magnitude of acute apoptotic cell death. This finding may be explained in part by changes in the Bcl-2:Bax ratio after a single binge EtOH exposure.

Time-dependent changes in gene expression induced by secreted amyloid precursor protein-alpha in the rat hippocampus.

BACKGROUND: Differential processing of the amyloid precursor protein liberates either amyloid-ß, a causative agent of Alzheimer's disease, or secreted amyloid precursor protein-alpha (sAPPα), which promotes neuroprotection, neurotrophism, neurogenesis and synaptic plasticity. The underlying molecular mechanisms recruited by sAPPα that underpin these considerable cellular effects are not well elucidated. As these effects are enduring, we hypothesised that regulation of gene expression may be of importance and examined temporally specific gene networks and pathways induced by sAPPα in rat hippocampal organotypic slice cultures. Slices were exposed to 1 nM sAPPα or phosphate buffered saline for 15 min, 2 h or 24 h and sAPPα-associated gene expression profiles were produced for each time-point using Affymetrix Rat Gene 1.0 ST arrays (moderated t-test using Limma: p < 0.05, and fold change ± 1.15). RESULTS: Treatment of organotypic hippocampal slice cultures with 1 nM sAPPα induced temporally distinct gene expression profiles, including mRNA and microRNA associated with Alzheimer's disease. Having demonstrated that treatment with human recombinant sAPPα was protective against N-methyl d-aspartate-induced toxicity, we next explored the sAPPα-induced gene expression profiles. Ingenuity Pathway Analysis predicted that short-term exposure to sAPPα elicited a multi-level transcriptional response, including upregulation of immediate early gene transcription factors (AP-1, Egr1), modulation of the chromatin environment, and apparent activation of the constitutive transcription factors CREB and NF-κB. Importantly, dynamic regulation of NF-κB appears to be integral to the transcriptional response across all time-points. In contrast, medium and long exposure to sAPPα resulted in an overall downregulation of gene expression. While these results suggest commonality between sAPPα and our previously reported analysis of plasticity-related gene expression, we found little crossover between these datasets. The gene networks formed following medium and long exposure to sAPPα were associated with inflammatory response, apoptosis, neurogenesis and cell survival; functions likely to be the basis of the neuroprotective effects of sAPPα. CONCLUSIONS: Our results demonstrate that sAPPα rapidly and persistently regulates gene expression in rat hippocampus. This regulation is multi-level, temporally specific and is likely to underpin the neuroprotective effects of sAPPα.

MicroRNAs in Parkinson's disease: a systematic review and diagnostic accuracy meta-analysis.

Current clinical tests for Parkinson's disease (PD) provide insufficient diagnostic accuracy leading to an urgent need for improved diagnostic biomarkers. As microRNAs (miRNAs) are promising biomarkers of various diseases, including PD, this systematic review and meta-analysis aimed to assess the diagnostic accuracy of biofluid miRNAs in PD. All studies reporting data on miRNAs expression in PD patients compared to controls were included. Gene targets and significant pathways associated with miRNAs expressed in more than 3 biofluid studies with the same direction of change were analyzed using target prediction and enrichment analysis. A bivariate model was used to calculate sensitivity, specificity, likelihood ratios, and diagnostic odds ratio. While miR-24-3p and miR-214-3p were the most reported miRNA (7 each), miR-331-5p was found to be consistently up regulated in 4 different biofluids. Importantly, miR-19b-3p, miR-24-3p, miR-146a-5p, and miR-221-3p were reported in multiple studies without conflicting directions of change in serum and bioinformatic analysis found the targets of these miRNAs to be associated with pathways important in PD pathology. Of the 102 studies from the systematic review, 15 studies reported sensitivity and specificity data on combinations of miRNAs and were pooled for meta-analysis. Studies (17) reporting sensitivity and specificity data on single microRNA were pooled in a separate meta-analysis. Meta-analysis of the combinations of miRNAs (15 studies) showed that biofluid miRNAs can discriminate between PD patients and controls with good diagnostic accuracy (sensitivity = 0.82, 95% CI 0.76-0.87; specificity = 0.80, 95% CI 0.74-0.84; AUC = 0.87, 95% CI 0.83-0.89). However, we found multiple studies included more males with PD than any other group therefore possibly introducing a sex-related selection bias. Overall, our study captures key miRNAs which may represent a point of focus for future studies and the development of diagnostic panels whilst also highlighting the importance of appropriate study design to develop representative biomarker panels for the diagnosis of PD.

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-CaMKII(T286)) 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.

A method for simultaneous detection of small and long RNA biotypes by ribodepleted RNA-Seq.

RNA sequencing offers unprecedented access to the transcriptome. Key to this is the identification and quantification of many different species of RNA from the same sample at the same time. In this study we describe a novel protocol for simultaneous detection of coding and non-coding transcripts using modifications to the Ion Total RNA-Seq kit v2 protocol, with integration of QIASeq FastSelect rRNA removal kit. We report highly consistent sequencing libraries can be produced from both frozen high integrity mouse hippocampal tissue and the more challenging post-mortem human tissue. Removal of rRNA using FastSelect was extremely efficient, resulting in less than 1.5% rRNA content in the final library. We identified > 30,000 unique transcripts from all samples, including protein-coding genes and many species of non-coding RNA, in biologically-relevant proportions. Furthermore, the normalized sequencing read count for select genes significantly negatively correlated with Ct values from qRT-PCR analysis from the same samples. These results indicate that this protocol accurately and consistently identifies and quantifies a wide variety of transcripts simultaneously. The highly efficient rRNA depletion, coupled with minimized sample handling and without complicated and high-loss size selection protocols, makes this protocol useful to researchers wishing to investigate whole transcriptomes.

Plasma microRNA vary in association with the progression of Alzheimer's disease.

INTRODUCTION: Early intervention in Alzheimer's disease (AD) requires the development of an easily administered test that is able to identify those at risk. Focusing on microRNA robustly detected in plasma and standardizing the analysis strategy, we sought to identify disease-stage specific biomarkers. METHODS: Using TaqMan microfluidics arrays and a statistical consensus approach, we assessed plasma levels of 185 neurodegeneration-related microRNA, in cohorts of cognitively normal amyloid ß-positive (CN-Aß+), mild cognitive impairment (MCI), and Alzheimer's disease (AD) participants, relative to their respective controls. RESULTS: Distinct disease stage microRNA biomarkers were identified, shown to predict membership of the groups (area under the curve [AUC] >0.8) and were altered dynamically with AD progression in a longitudinal study. Bioinformatics demonstrated that these microRNA target known AD-related pathways, such as the Phosphoinositide 3-kinase (PI3K-Akt) signalling pathway. Furthermore, a significant correlation was found between miR-27a-3p, miR-27b-3p, and miR-324-5p and amyloid beta load. DISCUSSION: Our results show that microRNA signatures alter throughout the progression of AD, reflect the underlying disease pathology, and may prove to be useful diagnostic markers.

Rapidly induced gene networks following induction of long-term potentiation at perforant path synapses in vivo.

The canonical view of the maintenance of long-term potentiation (LTP), a widely accepted experimental model for memory processes, is that new gene transcription contributes to its consolidation; however, the gene networks involved are unknown. To address this issue, we have used high-density Rat 230.2 Affymetrix arrays to establish a set of genes induced 20-min post-LTP, and using Ingenuity Pathway network analysis tools we have investigated how these early responding genes are interrelated. This analysis identified LTP-induced regulatory networks in which the transcription factors (TFs) nuclear factor-KB and serum response factor, which, to date, have not been widely recognized as coordinating the early gene response, play a key role alongside the more well-known TFs cyclic AMP response element-binding protein, and early growth response 1. Analysis of gene-regulatory promoter sites and chromosomal locations of the genes within the dataset reinforced the importance of these molecules in the early gene response and predicted that the coordinated action might arise from gene clustering on particular chromosomes. We have also identified a transcription-based response that affects mitogen-activated protein kinase signaling pathways and protein synthesis during the stabilization of the LTP response. Furthermore, evidence from biological function, networks, and regulatory analyses showed convergence on genes related to development, proliferation, and neurogenesis, suggesting that these functions are regulated early following LTP induction. This raises the interesting possibility that LTP-related gene expression plays a role in both synaptic reorganization and neurogenesis.

Astrocytic MicroRNA in Ageing, Inflammation, and Neurodegenerative Disease.

Astrocytes actively regulate numerous cell types both within and outside of the central nervous system in health and disease. Indeed, astrocyte morphology, gene expression and function, alongside the content of astrocyte-derived extracellular vesicles (ADEVs), is significantly altered by ageing, inflammatory processes and in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. Here, we review the relevant emerging literature focussed on perturbation in expression of microRNA (miRNA), small non-coding RNAs that potently regulate gene expression. Synthesis of this literature shows that ageing-related processes, neurodegenerative disease-associated mutations or peptides and cytokines induce dysregulated expression of miRNA in astrocytes and in some cases can lead to selective incorporation of miRNA into ADEVs. Analysis of the miRNA targets shows that the resulting downstream consequences of alterations to levels of miRNA include release of cytokines, chronic activation of the immune response, increased apoptosis, and compromised cellular functioning of both astrocytes and ADEV-ingesting cells. We conclude that perturbation of these functions likely exacerbates mechanisms leading to neuropathology and ultimately contributes to the cognitive or motor symptoms of neurodegenerative diseases. This field requires comprehensive miRNA expression profiling of both astrocytes and ADEVs to fully understand the effect of perturbed astrocytic miRNA expression in ageing and neurodegenerative disease.

Secreted Amyloid Precursor Protein-Alpha Enhances LTP Through the Synthesis and Trafficking of Ca2+-Permeable AMPA Receptors.

Regulation of AMPA receptor expression by neuronal activity and neuromodulators is critical to the expression of both long-term potentiation (LTP) and memory. In particular, Ca2+-permeable AMPARs (CP-AMPAR) play a unique role in these processes due to their transient, activity-regulated expression at synapses. Secreted amyloid precursor protein-alpha (sAPPα), a metabolite of the parent amyloid precursor protein (APP) has been previously shown to enhance hippocampal LTP as well as memory formation in both normal animals and in Alzheimer's disease models. In earlier work we showed that sAPPα promotes trafficking of GluA1-containing AMPARs to the cell surface and specifically enhances synthesis of GluA1. To date it is not known whether de novo synthesized GluA1 form CP-AMPARs or how they contribute to sAPPα-mediated plasticity. Here, using fluorescent non-canonical amino acid tagging-proximity ligation assay (FUNCAT-PLA), we show that brief treatment of primary rat hippocampal neurons with sAPPα (1 nM, 30 min) rapidly enhanced the cell-surface expression of de novo GluA1 homomers and reduced levels of de novo GluA2, as well as extant GluA2/3-AMPARs. The de novo GluA1-containing AMPARs were localized to extrasynaptic sites and later internalized by sAPPα-driven expression of the activity-regulated cytoskeletal-associated protein, Arc. Interestingly, longer exposure to sAPPα increased synaptic levels of GluA1/2 AMPARs. Moreover, the sAPPα-mediated enhancement of LTP in area CA1 of acute hippocampal slices was dependent on CP-AMPARs. Together, these findings show that sAPPα engages mechanisms which specifically enhance the synthesis and cell-surface expression of GluA1 homomers, underpinning the sAPPα-driven enhancement of synaptic plasticity in the hippocampus.

Increased expression, but not postsynaptic localisation, of ionotropic glutamate receptors during the late-phase of long-term potentiation in the dentate gyrus in vivo.

Long-term potentiation (LTP) is extensively studied as a cellular mechanism of information storage in the brain. The induction and early expression mechanisms of LTP depend on activation and rapid modulation of ionotropic glutamate receptors. However, the mechanisms that underlie maintenance of LTP over the course of days or longer are poorly understood. Here, we have investigated the overall expression of AMPA- and NMDA-type glutamate receptors (AMPARs and NMDARs, respectively), as well as their levels at the synaptic surface membrane and in the postsynaptic density (PSD), in the dentate gyrus at 48h following the induction of LTP at perforant path synapses in awake rats. We found a high-frequency stimulation-dependent increase in the overall levels of AMPAR subunits GluA1 and GluA2, but not GluA3 in the dentate gyrus. The increases in GluA1 and GluA2 levels were partially NMDAR-dependent, but were not found in biochemically isolated synaptic surface membrane or PSD fractions. In contrast, we found that the core NMDAR subunit, GluN1, increased in the synaptic surface-membrane fraction but it also was not targeted to the PSD. The GluA1 and GluA2 expression and the surface localisation of GluN1 returned to baseline levels by 2 weeks post-LTP induction. These data suggest that the late-phase LTP is not mediated by an overt increase in the AMPAR content of perforant path synapses. The increase in surface expression NMDARs may influence thresholds for future plasticity events.

Novel microRNA revealed by systematic analysis of the microRNA transcriptome in dentate gyrus granule cells.

Post-transcriptional control of gene expression by microRNAs provides an important regulatory system within neurons, allowing co-ordinate and fine-tuned expression of plasticity-related proteins. Indeed, specific microRNAs have been shown to be regulated by synaptic activity in the dentate gyrus, and contribute to the regulated gene expression that underlies the persistence of long-term potentiation (LTP), a model of memory. To fully explore the contribution of microRNAs in synaptic plasticity, it is important to characterize the complete microRNA transcriptome in regions such as the dentate gyrus. Accordingly we used deep sequencing and miRDeep* analysis to search for novel microRNAs expressed in the dentate gyrus granule cell layer. Drawing on combined sequencing and bioinformatics analyses, including hairpin stability and patterns of precursor microRNA processing, we identified nine putative novel microRNAs. We did not find evidence of differential expression of any of these putative microRNAs following LTP at perforant path-granule cell synapses in awake rats (5 h post-tetanus; p > 0.05). Focusing on novel_miR-1, the most abundant novel miRNA, we showed that this sequence could be amplified from RNA extracted from dentate gyrus granule cells by reverse transcription-quantitative polymerase chain reaction. Further, by computationally predicting mRNA targets of this microRNA, we found that this novel microRNA likely contributes to the regulation of proteins that function at synapses.

Plasma MicroRNAs Are Altered Early and Consistently in a Mouse Model of Tauopathy.

Pathological accumulation of tau protein in brain cells is the hallmark of a group of neurodegenerative diseases called tauopathies. Accumulation of tau protein begins years before the onset of symptoms, which include deficits in cognition, behavior and movement. The pre-symptomatic phase of tauopathy may be the best time to deliver disease-modifying treatments, but this is only possible if prognostic, pre-symptomatic biomarkers are identified. Here we describe the profiling of blood plasma microRNAs in a mouse model of tauopathy, in order to identify biomarkers of pre-symptomatic tauopathy. Circulating RNAs were isolated from blood plasma of 16-week-old and 53-week-old hTau mice and age-matched wild type controls (n = 28). Global microRNA profiling was performed using small RNA sequencing (Illumina) and selected microRNAs were validated using individual TaqMan RT-qPCR. The area under the receiver operating characteristic curve (AUC) was used to evaluate discriminative accuracy. We identified three microRNAs (miR-150-5p, miR-155-5p, miR-375-3p) that were down-regulated in 16-week-old hTau mice, which do not yet exhibit a behavioral phenotype and therefore represent pre-symptomatic tauopathy. The discriminative accuracy was AUC 0.98, 0.95 and 1, respectively. Down-regulation of these microRNAs persisted at 53 weeks of age, when hTau mice exhibit cognitive deficits and advanced neuropathology. Bioinformatic analysis showed that these three microRNAs converge on pathways associated with neuronal signaling and phosphorylation of tau. Thus, these circulating microRNAs appear to reflect neuropathological change and are promising candidates in the development of biomarkers of pre-symptomatic tauopathy.

Secreted Amyloid Precursor Protein-Alpha Promotes Arc Protein Synthesis in Hippocampal Neurons.

Secreted amyloid precursor protein-α (sAPPα) is a neuroprotective and memory-enhancing molecule, however, the mechanisms through which sAPPα promotes these effects are not well understood. Recently, we have shown that sAPPα enhances cell-surface expression of glutamate receptors. Activity-related cytoskeletal-associated protein Arc (Arg3.1) is an immediate early gene capable of modulating long-term potentiation, long-term depression and homeostatic plasticity through regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor localization. Accordingly, we hypothesized that sAPPα may enhance synaptic plasticity, in part, by the de novo synthesis of Arc. Using primary cortical and hippocampal neuronal cultures we found that sAPPα (1 nM, 2 h) enhances levels of Arc mRNA and protein. Arc protein levels were increased in both the neuronal somata and dendrites in a Ca2+/calmodulin-dependent protein kinase II-dependent manner. Additionally, dendritic Arc expression was dependent upon activation of mitogen-activated protein kinase and protein kinase G. The enhancement of dendritic Arc protein was significantly reduced by antagonism of N-methyl-D-aspartate (NMDA) and nicotinic acetylcholine (α7nACh) receptors, and fully eliminated by dual application of these antagonists. This effect was further corroborated in area CA1 of acute hippocampal slices. These data suggest sAPPα-regulated plasticity within hippocampal neurons is mediated by cooperation of NMDA and α7nACh receptors to engage a cascade of signal transduction molecules to enhance the transcription and translation of Arc.

Dopamine D1/D5 receptor activation reverses NMDA receptor-dependent long-term depression in rat hippocampus.

Activation of dopamine D1/D5 receptors (D1/D5Rs) in area CA1 of the rat hippocampus modulates the expression of synaptic plasticity in a manner that is dependent on the timing of the D1/D5R activation. Here, we measured field EPSPs in rat hippocampal slices to examine the modulation of long-term depression (LTD) in CA1 by D1/D5Rs when activated immediately after the induction of LTD by low-frequency stimulation (LFS) or bath application of NMDA or the metabotropic glutamate receptor agonist DHPG [(RS)-3,5-dihydroxyphenylglycine]. Activation of D1/D5Rs by SKF 38393 [(+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide] completely reversed a moderate LFS-induced LTD in a time-dependent manner, presumably through an adenylate cyclase/cAMP cascade. In support of this, general adenylate cyclase activation by forskolin ([3R-(3 alpha,4a beta,5 beta,6 beta,6a alpha,10 alpha,10a beta,10b alpha)]-5-(acetyloxy)-3-ethenyldodecahydro-6,10,10b-trihydroxy-3,4a,7,7,10a-pentamenthyl-1H-naphtho[2,1-b]pyran-1-one) immediately, but not 60 min, after LFS also reversed the LTD. Beta-adrenergic receptor activation by isoproterenol failed to reverse the LTD, indicating that reversal is specific to D1/D5R-mediated increased cAMP production. SKF 38393 only partially reversed a more robust LFS-induced LTD, indicating that some components of consolidated LTD are resistant to reversal. LTD induced by bath application of NMDA, but not DHPG, was also reversed by SKF 38393. Western blot analysis of postsynaptic density fractions after NMDA-induced LTD revealed that the LTD was attributable to dephosphorylation of the AMPA receptor subunit glutamate receptor 1 (GluR1) at serine 845, without a change in total GluR content. Reversal of the LTD by SKF 38393 was associated with rephosphorylation of this same residue. Together, these findings demonstrate a new role for dopamine in the neuromodulation of hippocampal LTD.

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.

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.

Bridging Synaptic and Epigenetic Maintenance Mechanisms of the Engram.

How memories are maintained, and how memories are lost during aging or disease, are intensely investigated issues. Arguably, the reigning theory is that synaptic modifications allow for the formation of engrams during learning, and sustaining engrams sustains memory. Activity-regulated gene expression profiles have been shown to be critical to these processes, and their control by the epigenome has begun to be investigated in earnest. Here, we propose a novel theory as to how engrams are sustained. We propose that many of the genes that are currently believed to underlie long-term memory are actually part of a "plasticity transcriptome" that underpins structural and functional modifications to neuronal connectivity during the hours to days following learning. Further, we hypothesize that a "maintenance transcriptome" is subsequently induced that includes epigenetic negative regulators of gene expression, particularly histone deacetylases. The maintenance transcriptome negatively regulates the plasticity transcriptome, and thus the plastic capability of a neuron, after learning. In this way, the maintenance transcriptome would act as a metaplasticity mechanism that raises the threshold for change in neurons within an engram, helping to ensure the connectivity is stabilized and memory is maintained.