Bruce G. Mockett

NMDA receptor-mediated metaplasticity during the induction of long-term depression by low-frequency stimulation

Metaplasticity refers to the activity‐dependent modification of the ability of synapses to undergo subsequent synaptic plasticity. Here, we have addressed the question of whether metaplasticity contributes to the induction of long‐term depression (LTD) by low‐frequency stimulation (LFS). The experiments were conducted using standard extracellular recording techniques in stratum radiatum of area CA1 in hippocampal slices made from adult Sprague–Dawley rats. The degree of LTD induction was found to be a nonlinear function of the number of pulses during a 1‐Hz LFS. Little LTD was observed following 600 or 900 pulses, but a significant LTD occurred following 1200 pulses of LFS, whether delivered in one episode, or in two bouts of 600 pulses given 10 min apart. A similar pattern was observed for 3 Hz LFS. The data support the suggestion that pulses occurring early in the LFS train prime synapses for LTD induction, as triggered by later occurring stimuli. The priming effect lasted at least 120 min, when tested by giving two bouts of 1 Hz LFS (600 pulses each) at different intervals. Neither heterosynaptic nor homosynaptic stimulation by itself was sufficient to prime LTD. However, a combination of the stimuli, induced by increased stimulus strength during the LFS, appeared necessary for inducing the effect. An N‐methyl‐d‐aspartate (NMDA) receptor antagonist markedly reduced total LTD induction, regardless of whether it was administered during the first or second LFS in a protocol employing two bouts of 600 pulse LFS, 30 min apart. These findings strongly support the hypothesis that NMDA receptor‐dependent metaplasticity processes contribute to the induction of LTD during standard LFS protocols.

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.

Dopamine D1/D5 receptor activation fails to initiate an activity-independent late-phase LTP in rat hippocampus.

The role of dopamine in the hippocampus remains poorly defined. Numerous studies have suggested that it acts as a neuromodulator of late-phase long-term potentiation (L-LTP) in CA1, while other reports controversially indicate that D1/D5 receptor (D1/D5R) activation may directly initiate activity-independent LTP. We have further investigated this putative role of dopamine in area CA1 in rat hippocampal slices using field potential recording techniques. Application of the dopamine D1/D5 receptor agonists SKF 38393 and 6-bromo-APB at 100 microM for 20 min did not induce an activity-independent L-LTP. Varying the incubation conditions still did not permit either SKF 38393 or an alternative D1/D5R agonist, 6-chloro-PB, to induce L-LTP. To further determine if intracellular mechanisms, which may act to limit the expression of LTP, were preventing D1/D5R-induced L-LTP expression, we inhibited protein phosphatase 1 activity by reducing cyclin-dependent kinase 5 (cdk5) inhibition of inhibitor 1. Inhibition of cdk5 by roscovitine (10 microM, 40 min) did not facilitate the ability of SKF 38393 to induce L-LTP in CA1. Biochemical experiments confirmed that the concentration of agonist used significantly elevated intracellular cAMP levels, suggesting that effective D1/D5R activation was achieved. Furthermore, coactivation with NMDA receptors (NMDAR) resulted in a synergistic increase in cAMP. These findings demonstrate that D1/D5R activation in CA1 initiates intracellular second messenger accumulation, but that this is insufficient to induce an activity-independent L-LTP.

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-(3α,4aβ,5β,6β,6aα,10α,10aβ,10bα)]-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. β-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.

METAPLASTICITY: NEW INSIGHTS THROUGH ELECTROPHYSIOLOGICAL INVESTIGATIONS

The term synaptic plasticity describes the ability of excitatory synapses to undergo activity-driven long-lasting changes in the efficacy of basal synaptic transmission. This change may be expressed as a long-term potentiation (LTP) or as a long-term depression (LTD). Metaplasticity is a higher-order form of synaptic plasticity that regulates the expression of both LTP and LTD through processes that are initiated by cellular activity that precedes a later bout of plasticity-inducing synaptic activity. Activation by prior synaptic activity and later expression as a facilitation or inhibition of activity-dependent synaptic plasticity are fundamental properties of metaplasticity. The intracellular mechanisms which support metaplasticity appear to be closely linked to those of synaptic plasticity, hence there are significant technical challenges to overcome in order to elucidate those mechanisms specific to metaplasticity. This review will examine the progress in the characterization of metaplasticity over the last decade or so with a focus on findings gained using electrophysiological techniques. It will look at the techniques applied, the brain regions investigated and the knowledge gained from the application of a wide range of protocols designed to examine the influence of varied forms of prior synaptic activity on later, activity-induced, synaptic plasticity.

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

BackgroundDifferential 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).ResultsTreatment 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.ConclusionsOur 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.

Therapeutic Potential of Secreted Amyloid Precursor Protein APPsα

Cleavage of the amyloid precursor protein (APP) by α-secretase generates an extracellularly released fragment termed secreted APP-alpha (APPsα). Not only is this process of interest due to the cleavage of APP within the amyloid-beta sequence, but APPsα itself has many physiological properties that suggest its great potential as a therapeutic target. For example, APPsα is neurotrophic, neuroprotective, neurogenic, a stimulator of protein synthesis and gene expression, and enhances long-term potentiation (LTP) and memory. While most early studies have been conducted in vitro, effectiveness in animal models is now being confirmed. These studies have revealed that either upregulating α-secretase activity, acutely administering APPsα or chronic delivery of APPsα via a gene therapy approach can effectively treat mouse models of Alzheimer’s disease (AD) and other disorders such as traumatic head injury. Together these findings suggest the need for intensifying research efforts to harness the therapeutic potential of this multifunctional protein.

Altered plasma arginine metabolome precedes behavioural and brain arginine metabolomic profile changes in the APPswe/PS1ΔE9 mouse model of Alzheimer’s disease

While amyloid-beta (Aβ) peptides play a central role in the development of Alzheimer’s disease (AD), recent evidence also implicates altered metabolism of L-arginine in the pathogenesis of AD. The present study systematically investigated how behavioural function and the brain and plasma arginine metabolic profiles changed in a chronic Aβ accumulation model using male APPswe/PS1ΔE9 transgenic (Tg) mice at 7 and 13 months of age. As compared to their wild-type (WT) littermates, Tg mice displayed age-related deficits in spatial water maze tasks and alterations in brain arginine metabolism. Interestingly, the plasma arginine metabolic profile was markedly altered in 7-month Tg mice prior to major behavioural impairment. Receiver operating characteristic curve analysis revealed that plasma putrescine and spermine significantly differentiated between Tg and WT mice. These results demonstrate the parallel development of altered brain arginine metabolism and behavioural deficits in Tg mice. The altered plasma arginine metabolic profile that preceded the behavioural and brain profile changes suggests that there may be merit in an arginine-centric set of ante-mortem biomarkers for AD.

SECRETED AMYLOID PRECURSOR PROTEIN-ALPHA ENHANCES LONG-TERM POTENTIATION VIA PROTEIN SYNTHESIS- AND PROTEIN TRAFFICKING-DEPENDENT MECHANISMS

risk. Complex cognitive activity has the potential to promote an array of neuroplastic alterations, and the ageing brain also has capacity to benefit from engaging in complex mental tasks. On the other hand, a high cognitive demand can induce stress. Findings from epidemiological studies have revealed that lifestyle factors, including stress, modulate the progression of AD. Methods: In this study we examined the effect of environmental enrichment (EE) initiated in mid-life in a transgenic (Tg) AD mouse model (APP/ PS1) and healthy wildtype (Wt) mice. At 6 months of age, when the Tg mice have established Ab plaque pathology, mice were randomly assigned to one of three different housing conditions for 6 months. Mice were randomly allocated to standard housing (SH), EE (toys, tunnels, running wheel, larger exploratory space), or EE+. Mice assigned to EE+ were housed in EE, however also entered a novel environment weekly. Following the 6-month differential housing period, we analysed the effect of environmental stimulation on Ab pathological burden and blood corticosterone levels. Results: TgADmice housed in EE+ showed increased Ab plaque pathology (M 1⁄4 2.70%) compared to SH (M 1⁄4 1.51%) or EE (M 1⁄4 1.98%) mice (p 1⁄4 .02). In addition, they had significantly increased blood corticosterone levels (ng/mL) (SH M 1⁄4 124.59; EE M 1⁄4 133.03; EE+ M 1⁄4 216.29, p 1⁄4 .01). Interestingly, the Tg EE+ mice demonstrated no significant cognitive differences to those in SH or EE despite increases in Ab pathology and the stress marker corticosterone. In addition, healthy Wt mice in EE+ did not show this pattern of heightened corticosterone. Conclusions: Our data suggest that long-term complex environmental stimulation introduced later in life induces stress and exacerbates existing Ab neuropathology in AD. The findings indicate that this kind of chronic stress may be a significant contributing factor in the development and progression of AD, particularly for those susceptible to the disease.

203. Prevention of Alzheimer's-Like Symptoms Using Lentiviral-Mediated Secreted Amyloid Precursor Protein-Alpha Overexpression in a Transgenic Mouse Model

Alzheimers disease (AD) is a neurodegenerative disease that is associated with memory loss and poor cognition. Pathologically, the disease is characterised by amyloid plaques and neurofibrillary tangles. The abnormally high level of amyloid-β is thought to be due in part to an imbalance in amyloid precursor protein cleavage whereby there is an increase in amyloid-β production concomitant with a decrease in the production of secreted amyloid precursor protein-alpha (sAPPα). sAPPα is neuroprotective, supports neurogenesis and regulates learning and memory. In the current study, a lentiviral vector containing sAPPα and green fluorescent protein (GFP) cDNA, under the control of a synapsin promoter was injected bilaterally into the hippocampus of 4-month old male APPSwe/PS1ΔE9 transgenic mice at four sites per hemisphere. The two control groups consisted of B6/C3 wild-type and transgenic littermates injected with an control vector containing only GFP. At 12 months of age, animals underwent behavioural testing to examine hippocampus-dependent memory performance. There were no significant differences between groups in the open field and elevated plus maze tests of activity and anxiety. During acquisition of spatial memory in a water maze, transgenic controls showed poor learning, as the total distance to the platform (3.0 m±0.7) was significantly higher than that for wild-type controls (1.79 m±0.43). However, this impaired learning effect was prevented in the transgenic sAPPα group (1.38 m±0.18. Transgenic mice injected with control vector performed poorly at water maze probe trials testing spatial memory retention, and had to swim a lot further to reach the target (35.45 m±2.85); however, in transgenic mice treated with sAPPα (26.07 m±1.83) performance was significantly improved (p < 0.05). Moreover, transgenic-sAPPα animals performed similarly to wildtype animals (25.43 m±2.03). There were no significant differences between groups in a dry-land spatial memory task – the Barnes maze, or a contextual fear-conditioning test. These results indicate that the overexpression of sAPPα is persistent for at least up to 8 months, and could prevent Alzheimers-like behavioural deficits in an animal model.Supported by a grant from the Health Research Council of New Zealand.