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Introduction: Neuronal Ca2+ signals generated through the activation of Ca2+-induced Ca2+ release in response to activity-generated Ca2+ influx play a significant role in hippocampal synaptic plasticity, spatial learning, and memory. We and others have previously reported that diverse stimulation protocols, or different memory-inducing procedures, enhance the expression of endoplasmic reticulum-resident Ca2+ release channels in rat primary hippocampal neuronal cells or hippocampal tissue.Methods and Results: Here, we report that induction of long-term potentiation (LTP) by Theta burst stimulation protocols of the CA3-CA1 hippocampal synapse increased the mRNA and protein levels of type-2 Ryanodine Receptor (RyR2) Ca2+ release channels in rat hippocampal slices. Suppression of RyR channel activity (1 h preincubation with 20 μM ryanodine) abolished both LTP induction and the enhanced expression of these channels; it also promoted an increase in the surface expression of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR1 and GluR2 and caused a moderate but significant reduction of dendritic spine density. In addition, training rats in the Morris water maze induced memory consolidation, which lasted for several days after the end of the training period, accompanied by an increase in the mRNA levels and the protein content of the RyR2 channel isoform.Discussion: We confirm in this work that LTP induction by TBS protocols requires functional RyR channels. We propose that the increments in the protein content of RyR2 Ca2+ release channels, induced by LTP or spatial memory training, play a significant role in hippocampal synaptic plasticity and spatial memory consolidation.

Triclosan, a widely used industrial and household agent, is present as an antiseptic ingredient in numerous products of everyday use, such as toothpaste, cosmetics, kitchenware, and toys. Previous studies have shown that human brain and animal tissues contain triclosan, which has been found also as a contaminant of water and soil. Triclosan disrupts heart and skeletal muscle Ca2+ signaling, damages liver function, alters gut microbiota, causes colonic inflammation, and promotes apoptosis in cultured neocortical neurons and neural stem cells. Information, however, on the possible effects of triclosan on the function of the hippocampus, a key brain region for spatial learning and memory, is lacking. Here, we report that triclosan addition at low concentrations to hippocampal slices from male rats inhibited long-term potentiation but did not affect basal synaptic transmission or paired-pulse facilitation and modified the content or phosphorylation levels of synaptic plasticity-related proteins. Additionally, incubation of primary hippocampal cultures with triclosan prevented both the dendritic spine remodeling induced by brain-derived neurotrophic factor and the emergence of spontaneous oscillatory Ca2+ signals. Furthermore, intra-hippocampal injection of triclosan significantly disrupted rat navigation in the Oasis maze spatial memory task, an indication that triclosan impairs hippocampus-dependent spatial memory performance. Based on these combined results, we conclude that triclosan exerts highly damaging effects on hippocampal neuronal function in vitro and impairs spatial memory processes in vivo.

Amyloid β peptide oligomers (AβOs), toxic aggregates with pivotal roles in Alzheimer’s disease, trigger persistent and low magnitude Ca2+ signals in neurons. We reported previously that these Ca2+ signals, which arise from Ca2+ entry and subsequent amplification by Ca2+ release through ryanodine receptor (RyR) channels, promote mitochondrial network fragmentation and reduce RyR2 expression. Here, we examined if AβOs, by inducing redox sensitive RyR-mediated Ca2+ release, stimulate mitochondrial Ca2+-uptake, ROS generation and mitochondrial fragmentation, and also investigated the effects of the antioxidant N-acetyl cysteine (NAC) and the mitochondrial antioxidant EUK-134 on AβOs-induced mitochondrial dysfunction. In addition, we studied the contribution of the RyR2 isoform to AβOs-induced Ca2+ release, mitochondrial Ca2+ uptake and fragmentation. We show here that inhibition of NADPH oxidase type-2 prevented the emergence of RyR-mediated cytoplasmic Ca2+ signals induced by AβOs in primary hippocampal neurons. Treatment with AβOs promoted mitochondrial Ca2+ uptake and increased mitochondrial superoxide and hydrogen peroxide levels; ryanodine, at concentrations that suppress RyR activity, prevented these responses. The antioxidants NAC and EUK-134 impeded the mitochondrial ROS increase induced by AβOs. Additionally, EUK-134 prevented the mitochondrial fragmentation induced by AβOs, as previously reported for NAC and ryanodine. These findings show that both antioxidants, NAC and EUK-134, prevented the Ca2+-mediated noxious effects of AβOs on mitochondrial function. Our results also indicate that Ca2+ release mediated by the RyR2 isoform causes the deleterious effects of AβOs on mitochondrial function. Knockdown of RyR2 with antisense oligonucleotides reduced by about 50% RyR2 mRNA and protein levels in primary hippocampal neurons, decreased by 40% Ca2+ release induced by the RyR agonist 4-chloro-m-cresol, and significantly reduced the cytoplasmic and mitochondrial Ca2+ signals and the mitochondrial fragmentation induced by AβOs. Based on our results, we propose that AβOs-induced Ca2+ entry and ROS generation jointly stimulate RyR2 activity, causing mitochondrial Ca2+ overload and fragmentation in a feed forward injurious cycle. The present novel findings highlight the specific participation of RyR2-mediated Ca2+ release on AβOs-induced mitochondrial malfunction.

Recognition memory comprises recollection judgment and familiarity, two different processes that engage the hippocampus and the perirhinal cortex, respectively. Previous studies have shown that aged rodents display defective recognition memory and alterations in hippocampal synaptic plasticity. We report here that young rats efficiently performed at short-term (5 min) and long-term (24 h) hippocampus-associated object-location tasks and perirhinal cortex-related novel-object recognition tasks. In contrast, aged rats successfully performed the object-location and the novel-object recognition tasks only at short-term. In addition, aged rats displayed defective long-term potentiation (LTP) and enhanced long-term depression (LTD). Successful long-term performance of object-location but not of novel-object recognition tasks increased the protein levels of ryanodine receptor types-2/3 (RyR2/RyR3) and of IP3R1 in young rat hippocampus. Likewise, sustained LTP induction (1 h) significantly increased RyR2, RyR3 and IP3R1 protein levels in hippocampal slices from young rats. In contrast, LTD induction (1 h) did not modify the levels of these three proteins. Naïve (untrained) aged rats displayed higher RyR2/RyR3 hippocampal protein levels but similar IP3R1 protein content relative to young rats; these levels did not change following exposure to either memory recognition task or after LTP or LTD induction. The perirhinal cortex from young or aged rats did not display changes in the protein contents of RyR2, RyR3, and IP3R1 after exposure at long-term (24 h) to the object-location or the novel-object recognition tasks. Naïve aged rats displayed higher RyR2 channel oxidation levels in the hippocampus compared to naïve young rats. The RyR2/RyR3 up-regulation and the increased RyR2 oxidation levels exhibited by aged rat hippocampus are likely to generate anomalous calcium signals, which may contribute to the well-known impairments in hippocampal LTP and spatial memory that take place during aging.

Increased reactive oxygen species (ROS) generation and the ensuing oxidative stress contribute to Alzheimer’s disease pathology. We reported previously that amyloid-β peptide oligomers (AβOs) produce aberrant Ca2+ signals at sublethal concentrations and decrease the expression of type-2 ryanodine receptors (RyR2), which are crucial for hippocampal synaptic plasticity and memory. Here, we investigated whether the antioxidant agent astaxanthin (ATX) protects neurons from AβOs-induced excessive mitochondrial ROS generation, NFATc4 activation, and RyR2 mRNA downregulation. To determine mitochondrial H2O2 production or NFATc4 nuclear translocation, neurons were transfected with plasmids coding for HyperMito or NFATc4-eGFP, respectively. Primary hippocampal cultures were incubated with 0.1 μM ATX for 1.5 h prior to AβOs addition (500 nM). We found that incubation with ATX (≤10 μM) for ≤24 h was nontoxic to neurons, evaluated by the live/dead assay. Preincubation with 0.1 μM ATX also prevented the neuronal mitochondrial H2O2 generation induced within minutes of AβOs addition. Longer exposures to AβOs (6 h) promoted NFATc4-eGFP nuclear translocation and decreased RyR2 mRNA levels, evaluated by detection of the eGFP-tagged fluorescent plasmid and qPCR, respectively. Preincubation with 0.1 μM ATX prevented both effects. These results indicate that ATX protects neurons from the noxious effects of AβOs on mitochondrial ROS production, NFATc4 activation, and RyR2 gene expression downregulation.