Although animal models of Alzheimer's disease (AD) recapitulate β-amyloid-dependent hippocampal synaptic and cognitive dysfunctions, it is poorly understood how cortex-dependent remote memory stabilization following initial hippocampal coding is affected. Here, we systematically analyzed biophysical and behavioral phenotypes, including remote memory functions, of 5XFAD APP/PS1 transgenic mice containing five familial AD mutations. We found that 5XFAD mice show hippocampal dysfunctions as observed by reduced levels of baseline transmission and long-term potentiation at Schaffer collateral-CA1 synapses. Hippocampus-dependent memory tested 1 day after contextual fear conditioning was also impaired age-dependently in 5XFAD mice, as correlated with the onset of hippocampal synaptic failures. Importantly, remote memory stabilization during 30 days after training significantly declined in 5XFAD mice at time well before the onset of hippocampal dysfunctions. Our results indicate that 5XFAD mice provide a useful model system to investigate the mechanisms and therapeutic interventions for multiple synaptic and memory dysfunctions associated with AD.

Here we report exocytosis of zymogen granules, as examined by multiphoton excitation imaging in intact pancreatic acini. Cholecystokinin induces Ca 2+ oscillations that trigger exocytosis when the cytosolic Ca 2+ concentration exceeds 1 microM. Zymogen granules fused with the plasma membrane maintain their Omega-shaped profile for an average of 220 s and serve as targets for sequential fusion of granules that are located within deeper layers of the cell. This secondary exocytosis occurs as rapidly as the primary exocytosis and accounts for most exocytotic events. Granule-granule fusion does not seem to precede primary exocytosis, indicating that secondary fusion events may require a plasma-membrane factor. This sequential-replenishment mechanism of exocytosis allows the cell to take advantage of a large supply of fusion-ready granules without needing to transport them to the plasma membrane.

J. Neurochem. (2010) 10.1111/j.1471‐4159.2010.06608.x Abstract β‐Site amyloid precursor protein cleaving enzyme 1 (BACE1) initiates amyloid‐β (Aβ) generation that is central to the pathophysiology of Alzheimer’s disease (AD). Therefore, lowering Aβ levels by BACE1 manipulations represents a key therapeutic strategy, but it remains unclear whether partial inhibition of BACE1, as expected for AD treatments, can improve memory deficits. In this study, we used heterozygous BACE1 gene knockout (BACE1+/−) mice to evaluate the effects of partial BACE1 suppression on different types of synaptic and cognitive dysfunctions in Alzheimer’s transgenic mice (5XFAD model). We found that ∼50% BACE1 reductions rescued deficits of 5XFAD mice not only in hippocampus‐dependent memories as tested by contextual fear conditioning and spontaneous alternation Y‐maze paradigms but also in cortex‐dependent remote memory stabilization during 30 days after contextual conditioning. Furthermore, 5XFAD‐associated impairments in long‐term potentiation (a synaptic model of learning and memory) and declines in synaptic plasticity/learning‐related brain‐derived neurotrophic factor‐tyrosine kinase B signaling pathways were prevented in BACE1+/−·5XFAD mice. Finally, these improvements were related with reduced levels of β‐secretase‐cleaved C‐terminal fragment (C99), Aβ peptides and plaque burden in relevant brain regions of BACE1+/−·5XFAD mice. Therefore, our findings provide compelling evidence for beneficial effects of partially BACE1‐inhibiting approaches on multiple forms of functional defects associated with AD.

Individual exocytic events in intact adrenal medulla were visualized by two-photon extracellular polar-tracer imaging. Exocytosis of chromaffin vesicles often occurred in a sequential manner, involving first vesicles located at the cell periphery and then those present deeper within the cytoplasm. Sequential exocytosis occurred preferentially at regions of the plasma membrane facing the intercellular space. The compound vesicles swelled to more than five times their original volume and formed vacuolar exocytic lumens as a result of expansion of intravesicular gels and their confinement within the lumen by the fusion pore and the narrow intercellular space. Such luminal swelling greatly promoted sequential exocytosis. The SNARE protein SNAP25 rapidly migrated from the plasma membrane to the membrane of fused vesicles. These data indicate that vesicles present deeper within the cytoplasm can be fusion ready like those at the cell periphery, and that swelling of exocytic lumens promotes assembly of the fusion machinery. We suggest the existence of two molecular configurations for fusion-ready states in Ca 2 þ -dependent exocytosis.

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Alzheimer's disease (AD) is characterized by accumulation of amyloid-␤ peptide (A␤) in the brain regions that subserve memory and cognition. The amylin receptor is a potential target receptor for expression of the deleterious actions of soluble oligomeric A␤ species. We investigated whether the amylin receptor antagonist, AC253, neutralizes the depressant effects of A␤ 1-42 and human amylin on hippocampal long-term potentiation (LTP). Furthermore, we examined whether depressed levels of LTP observed in transgenic mice, which overexpress amyloid precursor protein (TgCRND8), could be restored with AC253. In mouse hippocampal brain slices, field EPSPs were recorded from the stratum radiatum layer of the CA1 area (cornu ammonis 1 region of the hippocampus) in response to electrical stimulation of Schaeffer collateral afferents. LTP was induced by 3-theta burst stimulation protocols. A␤ 1-42 (50 nM) and human amylin (50 nM), but not A␤ 42-1 (50 nM), depressed LTP evoked using both stimulation protocols. Preapplication of AC253 (250 nM) blocked A␤-and human amylin-induced reduction of LTP without affecting baseline transmission or LTP on its own. In contrast to wild-type controls, where robust LTP is observed, 6-to 12-month-old TgCRND8 mice show blunted LTP that is significantly enhanced by application of AC253. Our data demonstrate that the effects of A␤ 1-42 and human amylin on LTP are expressed via the amylin receptor, and moreover, blockade of this receptor increases LTP in transgenic mice that show increased brain amyloid burden. Amylin receptor antagonists could serve as potentially useful therapeutic agents in AD.

Various fluorescent probes were assessed for investigating intact islets of Langerhans using two-photon excitation imaging. Polar fluorescent tracers applied on the outside rapidly (within 3 min) penetrated deep into the islets via microvessels. Likewise, an adenovirus carrying a Ca 2؉ -sensitive green fluorescent protein mutant gene, yellow cameleon 2.1, was successfully transfected and enabled ratiometric cytosolic Ca 2؉ measurement of cells in the deep layers of the islets. Interestingly, FM1-43, which is lipophilic and does not permeate the plasma membrane, also rapidly reached deep cell layers of the islets. In contrast, lipophilic fluorescent probes that permeate the plasma membrane (for example, fura-2-acetoxymethyl and BODIPY-forskolin) accumulated in the superficial cell layers of the islets, even 30 min after application. Thus, two-photon excitation imaging of pancreatic islets is a promising method for clarifying signaling mechanisms of islet cells, particularly when it is combined with membraneimpermeable probes. In addition, our data suggest that membrane-permeable antagonists may affect only the superficial cell layers of islets, and so their negative effects should be interpreted with caution. Diabetes 51 (Suppl. 1):S25-S28, 2002

Accumulating evidence indicates the key role of ␣-calcium/calmodulin-dependent protein kinase II (␣CaMKII) in synaptic plasticity and learning, but it remains unclear how this kinase participates in the processing of memory extinction. Here, we investigated the mechanism by which ␣CaMKII may mediate extinction by using heterozygous knock-in mice with a targeted T286A mutation that prevents the autophosphorylation of this kinase (␣CaMKII T286A+/-). Remarkably, partial reduction of ␣CaMKII function due to the T286A +/-mutation prevented the development of extinction without interfering with initial hippocampus-dependent memory formation as assessed by contextual fear conditioning and the Morris water maze. It is hypothesized that the mechanism of extinction may differ depending on the interval at which extinction training is started, being more akin to "new learning" at longer intervals and "unlearning" or "erasure" at shorter intervals. Consistent with this hypothesis, we found that extinction conducted 24 h, but not 15 min, after contextual fear training showed spontaneous recovery (reappearance of extinguished freezing responses) 21 d following the extinction, representing behavioral evidence for new learning and unlearning mechanisms underlying extinction 24 h and 15 min post-training, respectively. Importantly, the ␣CaMKII T286A+/-mutation blocked new learning of contextual fear memory extinction, whereas it did not interfere with unlearning processes. Our results demonstrate a genetic dissociation of new learning and unlearning mechanisms of extinction, and suggest that ␣CaMKII is responsible for extinguishing memories specifically through new learning mechanisms.