An important step for cholinergic transmission involves the vesicular storage of acetylcholine (ACh), a process mediated by the vesicular acetylcholine transporter (VAChT). In order to understand the physiological roles of the VAChT, we developed a genetically altered strain of mice with reduced expression of this transporter. Heterozygous and homozygous VAChT knockdown mice have a 45% and 65% decrease in VAChT protein expression, respectively. VAChT deficiency alters synaptic vesicle filling and affects ACh release. Whereas VAChT homozygous mutant mice demonstrate major neuromuscular deficits, VAChT heterozygous mice appear normal in that respect and could be used for analysis of central cholinergic function. Behavioral analyses revealed that aversive learning and memory are not altered in mutant mice; however, performance in cognitive tasks involving object and social recognition is severely impaired. These observations suggest a critical role of VAChT in the regulation of ACh release and physiological functions in the peripheral and central nervous system.

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Coffee is a popular beverage consumed worldwide and its effect on health protection has been well studied throughout literature. This study investigates the effect of chronic coffee and caffeine ingestion on cognitive behavior and the antioxidant system of rat brains. The paradigms of open field and object recognition were used to assess locomotor and exploratory activities, as well as learning and memory. The antioxidant system was evaluated by determining the activities of glutathione reductase (GR), glutathione peroxidase (GPx) and superoxide dismutase (SOD), as well as the lipid peroxidation and reduced glutathione content. Five groups of male rats were fed for approximately 80 days with different diets: control diet (CD), fed a control diet; 3% coffee diet (3%Co) and 6% coffee diet (6%Co), both fed a diet containing brewed coffee; 0.04% caffeine diet (0.04%Ca) and 0.08% caffeine diet (0.08%Ca), both fed a control diet supplemented with caffeine. The estimated caffeine intake was approximately 20 and 40 mg/kg per day, for the 3%Co-0.04%Ca and 6%Co-0.08%Ca treatments, respectively. At 90 days of life, the animals were subjected to the behavioral tasks and then sacrificed. The results indicated that the intake of coffee, similar to caffeine, improved long-term memory when tested with object recognition; however, this was not accompanied by an increase in locomotor and exploratory activities. In addition, chronic coffee and caffeine ingestion reduced the lipid peroxidation of brain membranes and increased the concentration of reduced-glutathione. The activities of the GR and SOD were similarly increased, but no change in GPx activity could be observed. Thus, besides improving cognitive function, our data show that chronic coffee consumption modulates the endogenous antioxidant system in the brain. Therefore, chronic coffee ingestion, through the protection of the antioxidant system, may play an important role in preventing age-associated decline in the cognitive function.

Long-term memory (LTM) formation has been linked with functional strengthening of existing synapses and other processes including de novo synaptogenesis. However, it is unclear whether synaptogenesis can contribute to LTM formation. Here, using α-calcium/calmodulin kinase II autophosphorylation-deficient (T286A) mutants, we demonstrate that when functional strengthening is severely impaired, contextual LTM formation is linked with traininginduced PSD95 up-regulation followed by persistent generation of multiinnervated spines, a type of synapse that is characterized by several presynaptic terminals contacting the same postsynaptic spine. Both PSD95 up-regulation and contextual LTM formation in T286A mutants required signaling by the mammalian target of rapamycin (mTOR). Furthermore, we show that contextual LTM resists destabilization in T286A mutants, indicating that LTM is less flexible when synaptic strengthening is impaired. Taken together, we suggest that activation of mTOR signaling, followed by overexpression of PSD95 protein and synaptogenesis, contributes to formation of invariant LTM when functional strengthening is impaired.synaptic plasticity | hippocampus | immediate-early gene | reconsolidation A fundamental question in neuroscience concerns the cellular mechanisms that lead to long-term memory (LTM) formation (1-3). It has been shown that LTM formation is associated with and demands strengthening of existing synapses, socalled functional plasticity (3-5), but it may also be linked with a more overt form of structural plasticity, an increase in synapse density (6-9). It is, however, unclear whether de novo synaptogenesis is critical for LTM formation.The participation of synaptogenesis for LTM formation can be investigated when synaptic strengthening is fully blocked. It would be ideal to also block training-induced changes in neuronal excitability that have been implicated in memory formation (10). However, technically this block may not be achievable as, for example, there are distinct types of synaptic strengthening. As a proxy, here we have studied T286A missense mutant mice lacking autophosphorylation at threonine 286 of the α-isoform of calcium/calmodulindependent kinase II (αCaMKII), a major protein of glutamatergic synapses in the forebrain (11). The T286A mutants have fully blocked NMDA receptor-dependent synaptic strengthening at hippocampal CA1 synapses (11-14), and they have impaired regulation of the postburst afterhyperpolarization (AHP) in CA1 pyramidal neurons (15). Accordingly, T286A mutants are deficient in contextual fear conditioning after a single training trial (16). However, after five massed training trials, T286A mutants can form contextual LTM (16) and this type of LTM is hippocampus-dependent (14).Here, we have used T286A mutants as tools to investigate the mechanism of LTM formation when NMDA receptor-dependent synaptic strengthening is blocked. Our study provides evidence that activation of mTOR signaling in the hippocampus, followed by overexpression of PSD95 protein and...