Membrane‐tethered AKT kinase regulates basal synaptic transmission and early phase LTP expression by modulation of post‐synaptic AMPA receptor level

The serine/threonine kinase AKT/PKB plays a fundamental role in a wide variety of neuronal functions, including neuronal cell development, axonal growth, and synaptic plasticity. Multiple evidence link AKT signaling pathways to regulation of late phase long‐term synaptic plasticity, synaptogenesis, and spinogenesis, as well as long‐term memory formation. Nevertheless, the downstream effectors mediating the effects of AKT on early phase long‐term potentiation (eLTP) are currently unknown. Here we report that using different regimes of pharmacological activation and inhibition of AKT activity in acute hippocampal slices, we found that AKT regulates the post‐synaptic expression of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPA) receptors affecting solely the expression of eLTP, with no effect on its induction and maintenance. We further show that both maintenance of basal synaptic activity and expression of eLTP require plasma membrane tethering by activated AKT and that basal synaptic activity may be regulated via the direct effects of AKT1 on the expression level of post‐synaptic AMPA receptors bypassing the canonical AKT signaling. Finally, we establish that eLTP expression requires the involvement of both the canonical AKT signaling pathways and the direct effect of AKT1 on AMPA receptor activity/expression in the post‐synaptic membrane. © 2016 Wiley Periodicals, Inc.     

Role of potassium channels in Abeta(1-40)-activated apoptotic pathway in cultured cortical neurons

Potassium channel dysfunction has been implicated in Alzheimer's disease (AD). In the present study, by using potassium channel blocker tetraethylammonium (TEA), we investigated the relationship between the enhancement of potassium currents and the alteration of apoptotic cascade in the neuronal apoptotic model induced by beta-amyloid peptide 1-40(Abeta(1-40)). Cortical neurons exposed to Abeta(1-40) 5 muM developed a specific increase in the delayed rectifier potassium current (I(K)), but not the transient outward potassium currents (I(A)), before the appearance of neuronal apoptosis. Abeta(1-40) induced various apoptotic features such as chromatin condensation, a decrease in the amount of Bcl-2 protein, an increase in the amount of Bax protein, cytochrome c release from mitochondria, and caspase-3 activation. Potassium channel blocker 5 mM TEA attenuated Abeta(1-40)-induced neuronal death and prevented the alterations of all above mentioned apoptotic indicators. The study indicates that I(K) enhancement might play an important role in certain form of programmed cell death induced by beta-amyloid peptide (Abeta). Increased potassium channel activity might trigger the activation of apoptosis cascade in Abeta(1-40)-treated rat cortical neurons.     

Gamma-glutamylcysteine ethyl ester-induced up-regulation of glutathione protects neurons against Abeta(1-42)-mediated oxidative stress and neurotoxicity: implications for Alzheimer's disease

Glutathione (GSH) is an important endogenous antioxidant found in millimolar concentrations in the brain. GSH levels have been shown to decrease with aging. Alzheimer's disease (AD) is a neurodegenerative disorder associated with aging and oxidative stress. Abeta(1-42) has been shown to induce oxidative stress and has been proposed to play a central role in the oxidative damage detected in AD brain. It has been shown that administration of gamma-glutamylcysteine ethyl ester (GCEE) increases cellular levels of GSH, circumventing the regulation of GSH biosynthesis by providing the limiting substrate. In this study, we evaluated the protective role of up-regulation of GSH by GCEE against the oxidative and neurotoxic effects of Abeta(1-42) in primary neuronal culture. Addition of GCEE to neurons led to an elevated mean cellular GSH level compared with untreated control. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine (BSO) led to a 98% decrease in total cellular GSH compared with control, which was returned to control levels by addition of GCEE. Taken together, these results suggest that GCEE up-regulates cellular GSH levels which, in turn, protects neurons against protein oxidation, loss of mitochondrial function, and DNA fragmentation induced by Abeta(1-42). These results are consistent with the notion that up-regulation of GSH by GCEE may play a viable protective role in the oxidative and neurotoxicity induced by Abeta(1-42) in AD brain.     

Enhancement of AMPA currents and GluR1 membrane expression through PKA-coupled adenosine A(2A) receptors

Phosphorylation of glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by Protein Kinase A (PKA) is known to regulate AMPA receptor (AMPAR) trafficking and stabilization at the postsynaptic membrane, which in turn is one of the key mechanisms by which synaptic transmission and plasticity are tuned. However, not much is known as to how Gs-coupled receptors contribute to endogenous PKA-mediated regulation of AMPA receptor function. Here we report that activation of the excitatory A(2A) adenosine receptor by 2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680, 1-30 nM) facilitates AMPA-evoked currents in CA1 pyramidal neurons, by a mechanism dependent on PKA activation, but not on protein synthesis. This modulation of AMPA currents was mimicked by forskolin (1 μM) and did not occur in stratum radiatum interneurons. Superfusion of the A(2A) receptor agonist also caused an increase in the amplitude of miniature excitatory postsynaptic currents (mEPSCs), as well as in the membrane levels of GluR1 subunits phosphorylated at the PKA site (Ser845). The impact of this increase on GluR1-containing AMPA receptor expression was evidenced by the potentiation of LTP at the CA3-CA1 synapse that followed brief activation of A(2A) receptors. We thus propose that in conditions of increased adenosine availability, A(2A) receptor activation is responsible for setting part of the endogenous GluR1 Ser-845 phosphorylation tonus and hence, the availability of the GluR1-containing AMPA receptor extrasynaptic pool for synaptic insertion and reinforcement of synaptic strength.     

Plasma Abeta(1-40) and Abeta(1-42) and the risk of dementia: a prospective case-cohort study

BACKGROUND: Amyloid beta peptides (Abeta) are important components of plaques in Alzheimer's disease. Plasma concentrations of Abeta(1-40) and Abeta(1-42) rise with age and are increased in people with mutations that cause early-onset Alzheimer's disease. However, Abeta(1-42) concentrations may decrease early in the dementia process. We postulated that concentrations of Abeta(1-40) and Abeta(1-42) in plasma are associated with risk of dementia. METHODS: We did a case-cohort study embedded in the prospective, population-based Rotterdam Study. Of 6713 participants at risk for dementia, a random sample of 1756 people was drawn. During follow-up (mean 8.6 years), 392 incident dementia cases were identified. We investigated the association between plasma Abeta concentrations and risk of dementia and its subtypes using Cox proportional hazard models. FINDINGS: High concentrations of Abeta(1-40) but not Abeta(1-42) at baseline were associated with an increased risk of dementia. Compared with the first quartile of Abeta(1-40), age and sex-adjusted hazard ratios for dementia for the second, third, and fourth quartiles were 1.07 (95% CI 0.72-1.58), 1.16 (0.78-1.70), and 1.46 (1.01-2.12). People with an increased Abeta(1-42)/Abeta(1-40) ratio had a reduced risk of dementia. Compared with the first quartile of the Abeta(1-42)/Abeta(1-40) ratio, hazard ratios for the second, third, and fourth quartiles were 0.74 (0.53-1.02), 0.62 (0.44-0.88), and 0.47 (0.33-0.67). Associations were similar for Alzheimer's disease and vascular dementia. INTERPRETATION: High plasma concentrations of Abeta(1-40), especially when combined with low concentrations of Abeta(1-42), indicate an increased risk of dementia. A potential role of plasma Abeta concentrations as a marker of incipient dementia warrants further investigation.     

Proteomic identification of proteins oxidized by Abeta(1-42) in synaptosomes: implications for Alzheimer's disease

Protein oxidation has been implicated in Alzheimer's disease (AD) and can lead to loss of protein function, abnormal protein turnover, interference with cell cycle, imbalance of cellular redox potential, and eventually cell death. Recent proteomics work in our laboratory has identified specifically oxidized proteins in AD brain such as: creatine kinase BB, glutamine synthase, ubiquitin carboxy-terminal hydrolase L-1, dihydropyrimidase-related protein 2, alpha-enolase, and heat shock cognate 71, indicating that a number of cellular mechanisms are affected including energy metabolism, excitotoxicity and/or synaptic plasticity, protein turnover, and neuronal communication. Synapse loss is known to be an early pathological event in AD, and incubation of synaptosomes with amyloid beta peptide 1-42 (Abeta 1-42) leads to the formation of protein carbonyls. In order to test the involvement of Abeta(1-42) in the oxidation of proteins in AD brain, we utilized two-dimensional gel electrophoresis, immunochemical detection of protein carbonyls, and mass spectrometry to identify proteins from synaptosomes isolated from Mongolian gerbils. Abeta(1-42) treatment leads to oxidatively modified proteins, consistent with the notion that Abeta(1-42)-induced oxidative stress plays an important role in neurodegeneration in AD brain. In this study, we identified beta-actin, glial fibrillary acidic protein, and dihydropyrimidinase-related protein-2 as significantly oxidized in synaptosomes treated with Abeta(1-42). Additionally, H+-transporting two-sector ATPase, syntaxin binding protein 1, glutamate dehydrogenase, gamma-actin, and elongation factor Tu were identified as increasingly carbonylated. These results are discussed with respect to their potential involvement in the pathogenesis of AD.     

beta-amyloid (Abeta)42(43), abeta42, abeta40 and apoE immunostaining of plaques in fatal head injury

beta-Amyloid (Abeta) deposits are found in the brains of approximately one-third of patients who die within days after a severe head injury; their presence correlating strongly with possession of an apolipoprotein E (apoE)-epsilon4 allele. The aim of the study was to investigate the relationship between Abeta42, Abeta40 and apoE immunostaining of Abeta plaques in the cerebral cortex and the relevance of apoE genotype in 23 fatally head-injured patients. These cases were known to have Abeta deposits from a previous study in which they were examined and semiquantified and related to apoE genotype. In the present study, the temporal cortex was probed using four different antibodies that recognize Abeta42(43), Abeta40 and an antibody to apoE. Abeta42(43)-positive plaques were observed in all of the 23 cases and Abeta40 immunoreactivity in only 11 of the 23 cases. In addition, semiquantitative analysis showed that relatively fewer plaques were detected with anti-Abeta40 than anti-Abeta42(43). ApoE-immunoreactive plaques were identified in 18 of the 23 cases. The number of plaques stained for apoE was relatively less than for Abeta42(43) but greater than for Abeta40. Furthermore, the density of Abeta plaques detected using either Abeta42(43), Abeta40 or apoE antibodies was associated with possession of apoE-epsilon4 in an allele dose-dependent manner. The results are consistent with Abeta42(43) as the initially deposited species in brain parenchyma and provide evidence that apoE is involved in the early stages of amyloid deposition. Further, the findings may be of relevance to the role of apoE genotype in influencing outcome after acute brain injury.     

Low molecular weight thiol amides attenuate MAPK activity and protect primary neurons from Abeta(1-42) toxicity

Oxidative stress caused by various stimuli lead to oxidation of glutathione (GSH), the major redox power of the cell. Amyloid beta [Abeta(1-42)] is one of the key components of senile plaques and is involved in the progress initiation and triggers of Alzheimer's disease (AD). Lower GSH levels correlated with the activation of mitogen-activated proteins kinases (MAPK) have been demonstrated in AD, Parkinson's disease (PD) and other neurodegenerative disorders and have been proposed to play a central role in the deterioration of the aging and neurodegenerative brain. In this study, we evaluated the ability of low molecular weight thiol amides, N-acetyl cysteine amide (AD4) that replenishes GSH levels, N-acetyl glycine cysteine amide (AD7) and N-acetyl-Cys-Gly-Pro-Cys-amide (CB4) to protect primary neuronal culture against the oxidative and neurotoxic effects of Abeta(1-42) and to inhibit cisplatin- and hydrogen-peroxide-induced phosphorylation of two MAP kinases (MAPK), p38 and ERK1/2, in NIH3T3 cells. Cell death induced by Abeta(1-42) in primary neuronal cells was reversed by the thiol amides. Likewise, protein oxidation, loss of mitochondrial function and DNA fragmentation all returned to control levels by pretreatment with the three thiol amides. Elevated phosphorylation of ERK1/2 and p38 induced by cisplatin or H2O2 in NIH3T3 cells was lowered by AD4, AD7 and CB4 in a dose-dependent manner. Taken together, these results suggest that the thiol amides AD4, AD7 and CB4 protect neuronal cells against Abeta(1-42) toxicity by attenuating oxidative stress in correlation with inhibiting the MAPK phosphorylation cascade. These results are consistent with the notion that these small molecular thiol amides may play a viable protective role in the oxidative and neurotoxicity induced by Abeta(1-42) in AD brain.     

Receptor changes and LTP: an analysis using aniracetam, a drug that reversibly modifies glutamate (AMPA) receptors.

The hypothesis that long-term potentiation (LTP) involves receptor modifications was tested with aniracetam, a nootropic drug that selectively increases currents mediated by the AMPA subclass of glutamate receptors. Aniracetam had different effects on the waveform of synaptic potentials in hippocampus before and after induction of LTP: (1) the drug caused a slight reduction (or delay) of the initial segment of the response after LTP; and (2) the facilitatory effects of aniracetam occurred at a later time point in the response after LTP than before. The interactions between LTP and aniracetam were still present when synaptic responses were greatly reduced by partial blockade of postsynaptic receptors and were not reproduced by increasing release or the number of stimulated synapses. A mathematical treatment of synaptic currents produced the following results: (1) if aniracetam facilitates AMPA receptor currents simply by reducing desensitization, then its complex interaction with LTP emerges when potentiation changes the kinetic and conductance properties of receptor channels; (2) if aniracetam also significantly increases conductance, then the experimental data can be reproduced by modeling LTP as an increase in channel conductance alone.     

5-HT(1A) and 5-HT(7) receptors differently modulate AMPA receptor-mediated hippocampal synaptic transmission

We have studied the effects of 5-HT(1A) and 5-HT(7) serotonin receptor activation in hippocampal CA3-CA1 synaptic transmission using patch clamp on mouse brain slices. Application of either 5-HT or 8-OH DPAT, a mixed 5-HT(1A)/5-HT(7) receptor agonist, inhibited AMPA receptor-mediated excitatory post synaptic currents (EPSCs); this effect was mimicked by the 5-HT(1A) receptor agonist 8-OH PIPAT and blocked by the 5-HT(1A) antagonist NAN-190. 8-OH DPAT increased paired-pulse facilitation and reduced the frequency of mEPSCs, indicating a presynaptic reduction of glutamate release probability. In another group of neurons, 8-OH DPAT enhanced EPSC amplitude but did not alter paired-pulse facilitation, suggesting a postsynaptic action; this effect persisted in the presence of NAN-190 and was blocked by the 5-HT(7) receptor antagonist SB-269970. To confirm that EPSC enhancement was mediated by 5-HT(7) receptors, we used the compound LP-44, which is considered a selective 5-HT(7) agonist. However, LP-44 reduced EPSC amplitude in most cells and instead increased EPSC amplitude in a subset of neurons, similarly to 8-OH DPAT. These effects were respectively antagonized by NAN-190 and by SB-269970, indicating that under our experimental condition LP-44 behaved as a mixed agonist. 8-OH DPAT also modulated the current evoked by exogenously applied AMPA, inducing either a reduction or an increase of amplitude in distinct neurons; these effects were respectively blocked by 5-HT(1A) and 5-HT(7) receptor antagonists, indicating that both receptors exert a postsynaptic action. Our results show that 5-HT(1A) receptors inhibit CA3-CA1 synaptic transmission acting both pre- and postsynaptically, whereas 5-HT(7) receptors enhance CA3-CA1 synaptic transmission acting exclusively at a postsynaptic site. We suggest that a selective pharmacological targeting of either subtype may be envisaged in pathological loss of hippocampal-dependent cognitive functions. In this respect, we underline the need for new selective agonists of 5-HT(7) receptors.     

Early formation of mature amyloid-beta protein deposits in a mutant APP transgenic model depends on levels of Abeta(1-42).

The main objective of the present study was to develop an alternative singly-transgenic (tg) hAPP model where amyloid deposition will occur at an earlier age. For this purpose, we generated lines of tg mice expressing hAPP751 cDNA containing the London (V717I) and Swedish (K670M/N671L) mutations under the regulatory control of the murine (m)Thy-1 gene (mThy1-hAPP751). In the brains of the highest (line 41) and intermediate (lines 16 and 11) expressers, high levels of hAPP expression were found in neurons in layers 4-5 of the neocortex, hippocampal CA1 and olfactory bulb. As early as 3-4 months of age, line 41 mice developed mature plaques in the frontal cortex, whereas at 5-7 months plaque formation extended to the hippocampus, thalamus and olfactory region. Ultrastructural and double-immunolabeling analysis confirmed that most plaques were mature and contained dystrophic neurites immunoreactive with antibodies against APP, synaptophysin, neurofilament and tau. In addition, a decrease in the number of synaptophysin-immunoreactive terminals was most prominent in the frontal cortex of mice from line 41. Mice from line 11 developed diffuse amyloid deposits at 11 months of age, whereas mice from line 16 did not show evidence of amyloid deposition. Analysis of Abeta by ELISA showed that levels of Abeta(1-40) were higher in mice that did not show any amyloid deposits (line 16), whereas Abeta(1-42) was the predominant species in tg animals from the lines showing plaque formation (lines 41 and 11). Taken together this study indicates that early onset plaque formation depends on levels of Abeta(1-42).     

Acute stress impairs (or induces) synaptic long-term potentiation (LTP) but does not affect paired-pulse facilitation in the stratum radiatum of rat hippocampus.

Rats were exposed to restraint coupled with 60, 1-sec, 1-mA, 60-Hz tail shocks. One hippocampus was immediately dissected for in vitro measurement of paired-pulse facilitation and LTP of the excitatory postsynaptic potential (EPSP) recording from the stratum radiatum of field CA1. There was no change in paired-pulse facilitation, suggesting that acute exposure to the stressor does not result in a decrease in presynaptic neurotransmitter release. There was, however, a significant decrease in the percent LTP produced by theta burst stimulation relative to naive controls. These results are consistent with the hypothesis that the stress-induced impairment of LTP is a result of changes in the postsynaptic glutamate receptors, specifically the AMPA type.     

Abeta(1-40)-induced secretion of matrix metalloproteinase-9 results in sAPPalpha release by association with cell surface APP

To understand matrix metalloproteinase-9 (MMP-9) involvement in Alzheimer's disease, we examined mechanisms mediating increased expression of MMP-9 in the presence of Abeta(1-40) and the role of MMP-9 on amyloid precursor protein (APP) processing. Up-regulation of MMP-9 expressed by SK-N-SH cells in the presence of Abeta(1-40) was mediated by alpha(3)beta(1) and alpha(2)beta(1) integrin receptors. Overexpression of MMP-9 or treatment of HEK/APP695 cells with activated recombinant MMP-9 resulted in enhanced secretion of soluble APP (sAPPalpha), a product of alpha-secretase cleavage, and reduction of Abeta release. MMP-9 effect was enhanced by phorbol 12-mysistrate-13-acetate (PMA), an alpha-secretase activator and inhibited by EDTA or SB-3CT, an MMP-9 inhibitor. Additionally, immunoprecipitation and confocal microscopy demonstrated that MMP-9 and APP695 were associated on the cell surface. These results indicate that Abeta peptide increases MMP-9 secretion through integrins; MMP-9 then directly processes cell surface APP695 with an alpha-secretase like activity, substantially reducing the levels of secreted Abeta peptide.     

AGS-induced expression of Narp is concomitant with expression of AMPA receptor subunits GluR1 and GluR2 in hippocampus but not inferior colliculus of P77PMC rats

To explore mechanisms of epileptogenesis in audiogenic seizures (AGS), we examined the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleopropionic acid (AMPA) receptor subunits GluR1 and GluR2 and of the GluR-associated protein Narp in the hippocampus and the inferior colliculus (IC) from AGS-susceptible P77PMC rats after a single AGS and audiogenic kindling. Western blotting and immunohistochemistry showed that Narp was rapidly induced in both the hippocampus and the IC by AGS. In the hippocampus, up-regulation of Narp was concomitant with GluR1 and GluR2 under both conditions of a single AGS and AGS kindling. In the IC, however, Narp was up-regulated, GluR2 down-regulated, and GluR1 unchanged after kindling. In comparison with kindling, neither GluR1 nor GluR2 was changed, while Narp significantly increased in the IC following a single AGS. These findings suggest that down-regulation of AMPA receptor GluR2 subunit in the IC may contribute to AGS-mediated epileptogenesis, and up-regulation of Narp in the IC may be involved in audiogenic seizures.     

Abeta(1-42)-induced JNK and ERK activation in rabbit hippocampus is differentially regulated by lithium but is not involved in the phosphorylation of tau

Administration of Abeta(1-42) into the rabbit brain induces apoptosis and phosphorylation of tau. These Abeta effects correlate with the activation of JNK and ERK, but not of p38. Treatment with 7 mM lithium inhibits apoptosis, modulates JNK and ERK and does not affect the phosphorylation of tau. Our results demonstrate that lithium, at this dose, effectively inhibits the Abeta-induced apoptosis but has no effect on tau phosphorylation, and that MAP kinases are not involved in the phosphorylation of tau.     

Gamma-glutamylcysteine ethyl ester protection of proteins from Abeta(1-42)-mediated oxidative stress in neuronal cell culture: a proteomics approach

Protein oxidation mediated by amyloid beta-peptide (1-42) (Abeta[1-42]) has been proposed to play a central role in the pathogenesis of Alzheimer's disease (AD), a neurodegenerative disorder associated with aging and the loss of cognitive function. The specific mechanism by which Abeta(1-42), the primary component of the senile plaque and a pathologic hallmark of AD, contributes to the oxidative damage evident in AD brain is unknown. Moreover, the specific proteins that are vulnerable to oxidative damage induced by Abeta(1-42) are unknown. Identification of such proteins could contribute to our understanding of not only the role of Abeta(1-42) in the pathogenesis of AD, but also provide insight into the mechanisms of neurodegeneration at the protein level in AD. We report the proteomic identification of two proteins found to be oxidized significantly in neuronal cultures treated with Abeta(1-42): 14-3-3zeta and glyceraldehyde-3-phosphate dehydrogenase. We also report that pretreatment of neuronal cultures with gamma-glutamylcysteine ethyl ester, a compound that supplies the limiting substrate for the synthesis of glutathione and results in the upregulation of glutathione in neuronal cultures, protects both proteins against Abeta(1-42)-mediated protein oxidation.     

A presenilin 1 mutation (L420R) in a family with early onset Alzheimer disease,seizures and cotton wool plaques,but not spastic paraparesis

Over 100 mutations in the presenilin‐1 gene (PSEN1) have been shown to result in familial early onset Alzheimer disease (EOAD), but only a relatively few give rise to plaques with an appearance like cotton wool (CWP) and/or spastic paraparesis (SP). A family with EOAD, seizures and CWP was investigated by neuropathological study and DNA sequencing of the PSEN1 gene. Aβ was identified in leptomeningeal vessels and in cerebral plaques. A single point mutation, p.L420R (g.1508T > G) that gives rise to a missense mutation in the eighth transmembrane (TM8) domain of PS1 was identified in two affected members of the family. p.L420R (g.1508T > G) is the mutation responsible for EOAD, seizures and CWP without SP in this family.     

Selective scarcity of NMDA receptor channel subunits in the stratum lucidum (mossy fibre‐recipient layer) of the mouse hippocampal CA3 subfield

Hippocampal synapses express two distinct forms of the long-term potentiation (LTP), i.e. NMDA receptor-dependent and -independent LTPs. To understand its molecular-anatomical basis, we produced affinity-purified antibodies against the GluRε1 (NR2A), GluRε2 (NR2B), and GluRζ1 (NR1) subunits of the N-methyl-d -aspartate (NMDA) receptor channel, and determined their distributions in the mouse hippocampus. Using NMDA receptor subunit-deficient mice as the specificity controls, section pretreatment with proteases (pepsin and proteinase K) was found to be very effective to detect authentic NMDA receptor subunits. As the result of modified immunohistochemistry, all three subunits were detected at the highest level in the strata oriens and radiatum of the CA1 subfield, and high levels were also seen in most other neuropil layers of the CA1 and CA3 subfields and of the dentate gyrus. However, the stratum lucidum, a mossy fibre-recipient layer of the CA3 subfield, contained low levels of the GluRε1 and GluRζ1 subunits and almost excluded the GluRε2 subunit. Double immunofluorescence with the AMPA receptor GluRα1 (GluR1 or GluR-A) subunit further demonstrated that the GluRε1 subunit was colocalized in a subset, not all, of GluRα1-immunopositive structures in the stratum lucidum. Therefore, the selective scarcity of these NMDA receptor subunits in the stratum lucidum suggests that a different synaptic targeting mechanism exerts within a single CA3 pyramidal neurone in vivo, which would explain contrasting significance of the NMDA receptor channel in LTP induction mechanisms between the mossy fibre-CA3 synapse and other hippocampal synapses.