Aberrant Amygdala-Dependent Cued Fear Memory in Na+/Ca2+ Exchanger 1 Heterozygous Mice.

Na+/Ca2+ exchangers (NCXs) are mainly expressed in the plasma membrane and exchange one Ca2+ for three Na+, depending on the electrochemical gradients across the plasma membrane. NCXs have three isoforms, NCX1-3, encoded by distinct genes in mammals. Here, we report that heterozygous mice lacking NCX1 (NCX1+/-) exhibit impaired amygdala-dependent cued fear memory. NCX1+/- mice showed significant impairment in fear-related behaviors measured with the elevated-plus maze, light-dark, open-field, and marble-burying tasks. In addition, NCX1+/- mice showed abnormality in cued fear memory but not in contextual fear memory in a fear-conditioning task. In immunohistochemical analyses, NCX1+/- mice had significantly increased number of c-Fos-positive cells in the lateral amygdala (LA) but not in the central amygdala following fear-related tone stimuli. c-Fos expression peaked at 1 h. In concordance with the aberrant fear-related behaviors in NCX1+/- mice, enhanced long-term potentiation was also observed in the LA of these mice. Furthermore, enhancement of CaMKII or CaMKIV activity in the LA was observed in NCX1+/- mice by immunoblot analyses. In contrast, CaMKII+/- but not CaMKIV-/- mice insufficiently exhibited tone-induced cued fear memory and there was no increase in the number of c-Fos-positive cells in the LA. Altogether, the increased CaMKII activity and consequent c-Fos expression likely account for the dysregulation of amygdala-dependent cued fear memory in NCX1+/- mice.

Reduced expression of Na+/Ca2+ exchangers is associated with cognitive deficits seen in Alzheimer's disease model mice.

Na+/Ca2+ exchangers (NCXs) are expressed primarily in the plasma membrane of most cell types, where they mediate electrogenic exchange of one Ca2+ for three Na+ ions, depending on Ca2+ and Na+ electrochemical gradients across the membrane. Three mammalian NCX isoforms (NCX1, NCX2, and NCX3) are each encoded by a distinct gene. Here, we report that NCX2 and NCX3 protein and mRNA levels are relatively reduced in hippocampal CA1 of APP23 and APP-KI mice. Likewise, NCX2+/- or NCX3+/- mice exhibited impaired hippocampal LTP and memory-related behaviors. Moreover, relative to controls, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in NCX2+/- mouse hippocampus but increased in hippocampus of NCX3+/- mice. NCX2 or NCX3 heterozygotes displayed impaired maintenance of hippocampal LTP, a phenotype that in NCX2+/- mice was correlated with elevated calcineurin activity and rescued by treatment with the calcineurin (CaN) inhibitor FK506. Likewise, FK506 treatment significantly restored impaired hippocampal LTP in APP-KI mice. Moreover, Ca2+ clearance after depolarization following high frequency stimulation was slightly delayed in hippocampal CA1 regions of NCX2+/- mice. Electron microscopy revealed relatively decreased synaptic density in CA1 of NCX2+/- mice, while the number of spines with perforated synapses in CA1 significantly increased in NCX3+/- mice. We conclude that memory impairment seen in NCX2+/- and NCX3+/- mice reflect dysregulated hippocampal CaMKII activity, which alters dendritic spine morphology, findings with implications for memory deficits seen in Alzheimer's disease model mice.

Reduced CaM Kinase II and CaM Kinase IV Activities Underlie Cognitive Deficits in NCKX2 Heterozygous Mice.

Among five members of the K+-dependent Na+/Ca2+ exchanger (NCKX) family (NCKX1-5), only NCKX2 is highly expressed in mouse brain. NCKX2 in plasma membranes mediates cytosolic calcium excretion through electrogenic exchange of 4 Na+ for 1 Ca2+ and 1 K+. Here, we observed significantly decreased levels of NCKX2 protein and mRNA in the CA1 region of APP23 mice, a model of Alzheimer's disease. We also found that, like APP23 mice, heterozygous NCKX2-mutant mice exhibit mildly impaired hippocampal LTP and memory acquisition, the latter based on novel object recognition and passive avoidance tasks. When we addressed underlying mechanisms, we found that both CaMKII autophosphorylation and CaMKIV phosphorylation significantly decreased in CA1 regions of NCKX2+/- relative to control mice. Likewise, phosphorylation of GluA1 (Ser-831) and CREB (Ser-133), respective downstream targets of CaMKII and CaMKIV, also significantly decreased in the CA1 region. BDNF protein and mRNA levels significantly decreased in CA1 of NCKX2+/- relative to control mice. Finally, CaN activity increased in CA1 of NCKX2+/- mice. Our findings suggest that like APP23 mice, NCKX2+/- mice may exhibit impaired learning and hippocampal LTP due to decreased CaM kinase II and CaM kinase IV activities.

Aberrant behavioral sensitization by methamphetamine in junctophilin-deficient mice.

Junctophilins (JPs) expressed in the endoplasmic/sarcoplasmic reticulum (ER/SR) interact with the plasma membrane, thereby constructing junctional membrane complexes (JMC). We here reported that double-knockout mice lacking both JP3 and JP4 (JP-DKO mice) exhibit aberrant synaptic plasticity in the corticostriatal circuits and irregular methamphetamine (METH)-induced behavioral sensitization when METH (1.0 mg/kg) was administrated six consecutive days and assessed the striatal glutamatergic population spike (PS) by stimulation of cortical white matter. When we assessed the striatal PS by stimulation of cortical white matter, the long-term depression (LTD) was observed in JP-DKO mouse striatum similar to that in control (JP-double hetero mice (JP-DHE mice)). Importantly, LTD converted to long-term potentiation (LTP) following chronic METH treatment concomitant with behavioral sensitization in JP-DHE mice. LTD in JP-DKO mice, however failed to convert to LTP with lacks of behavioral sensitization. LTP impairment in JP-DKO mice was restored by pretreatment with FK506, calcineurin (CaN) inhibitor, but not with apamin, SK channel inhibitor. In immunoblotting analyses, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation was significantly increased following METH treatment in the striatum of JP-DHE mice. However, CaMKII autophosphorylation did not changed by METH treatment in the striatum of JP-DKO mouse. The increased CaMKII autophosphorylation was closely associated with elevated CaN activity in JP-DKO mice. The lack of increased CaMKII activity in JP-DKO mice was correlated with the impaired METH-induced behavioral sensitization. Thus, elevated CaN and aberrant CaMKII activities in the striatum of JP-DKO mice likely accounts for lack of METH-induced behavioral sensitization.

Stimulation of Sigma-1 Receptor Ameliorates Depressive-like Behaviors in CaMKIV Null Mice.

Sigma-1 receptor (Sig-1R) is a molecular chaperone regulating calcium efflux from the neuronal endoplasmic reticulum to the mitochondria. Calcium/calmodulin-dependent protein kinase IV (CaMKIV) null mice exhibit depressive-like behaviors and impaired neurogenesis as assessed by bromodeoxyuridine (BrdU) incorporation into newborn cells of the hippocampal dentate gyrus (DG). Here, we demonstrate that chronic stimulation of Sig-1R by treatment with the agonist SA4503 or the SSRI fluvoxamine for 14 days improves depressive-like behaviors in CaMKIV null mice. By contrast, treatment with paroxetine, which lacks affinity for Sig-1R, did not alter these behaviors. Reduced numbers of BrdU-positive cells and decreased brain-derived neurotrophic factor (BDNF) mRNA expression and protein kinase B (Akt; Ser-473) phosphorylation seen in the DG of CaMKIV null mice were significantly rescued by chronic Sig-1R stimulation. Interestingly, reduced ATP production observed in the DG of CaMKIV null mice was improved by chronic Sig-1R stimulation. Such stimulation also improved hippocampal long-term potentiation (LTP) induction and maintenance, which are impaired in the DG of CaMKIV null mice. LTP rescue was closely associated with both increases in calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and GluA1 (Ser-831) phosphorylation. Taken together, Sig-1R stimulation by SA4503 or fluvoxamine treatment increased hippocampal neurogenesis, which is closely associated with amelioration of depressive-like behaviors in CaMKIV null mice.

CaMKII activity is essential for improvement of memory-related behaviors by chronic rivastigmine treatment.

Because the cholinergic system is down-regulated in the brain of Alzheimer's disease patients, cognitive deficits in Alzheimer's disease patients are significantly improved by rivastigmine treatment. To address the mechanism underlying rivastigmine-induced memory improvements, we chronically treated olfactory bulbectomized (OBX) mice with rivastigmine. The chronic rivastigmine treatments for 12-13 days starting at 10 days after OBX operation significantly improved memory-related behaviors assessed by Y-maze task, novel object recognition task, passive avoidance task, and Barnes maze task, whereas the single rivastigmine treatment failed to improve the memory. Consistent with the improved memory-related behaviors, long-term potentiation in the hippocampal CA1 region was markedly restored by rivastigmine treatments. In immunoblotting analyses, the reductions of calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and calcium/calmodulin-dependent protein kinase IV (CaMKIV) phosphorylation in the CA1 region in OBX mice were significantly restored by rivastigmine treatments. In addition, phosphorylation of AMPAR subunit glutamate receptor 1 (GluA1) (Ser-831) and cAMP-responsive element-binding protein (Ser-133) as downstream targets of CaMKII and CaMKIV, respectively, in the CA1 region was also significantly restored by chronic rivastigmine treatments. Finally, we confirmed that rivastigmine-induced improvements of memory-related behaviors and long-term potentiation were not obtained in CaMKIIα(+/-) mice. On the other hand, CaMKIV(-/-) mice did not exhibit the cognitive impairments. Taken together, the stimulation of CaMKII activity in the hippocampus is essential for rivastigmine-induced memory improvement in OBX mice.

Stimulation of the sigma-1 receptor by DHEA enhances synaptic efficacy and neurogenesis in the hippocampal dentate gyrus of olfactory bulbectomized mice.

Dehydroepiandrosterone (DHEA) is the most abundant neurosteroid synthesized de novo in the central nervous system. We previously reported that stimulation of the sigma-1 receptor by DHEA improves cognitive function by activating calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C and extracellular signal-regulated kinase in the hippocampus in olfactory bulbectomized (OBX) mice. Here, we asked whether DHEA enhances neurogenesis in the subgranular zone of the hippocampal dentate gyrus (DG) and improves depressive-like behaviors observed in OBX mice. Chronic treatment with DHEA at 30 or 60 mg/kg p.o. for 14 days significantly improved hippocampal LTP impaired in OBX mice concomitant with increased CaMKII autophosphorylation and GluR1 (Ser-831) phosphorylation in the DG. Chronic DHEA treatment also ameliorated depressive-like behaviors in OBX mice, as assessed by tail suspension and forced swim tests, while a single DHEA treatment had no affect. DHEA treatment also significantly increased the number of BrdU-positive neurons in the subgranular zone of the DG of OBX mice, an increase inhibited by treatment with NE-100, a sigma-1 receptor antagonist. DHEA treatment also significantly increased phosphorylation of Akt (Ser-473), Akt (Ser-308) and ERK in the DG. Furthermore, GSK-3ß (Ser-9) phosphorylation increased in the DG of OBX mice possibly accounting for increased neurogenesis through Akt activation. Finally, we confirmed that DHEA treatment of OBX mice increases the number of BrdU-positive neurons co-expressing ß-catenin, a downstream GSK-3ßtarget. Overall, we conclude that sigma-1 receptor stimulation by DHEA ameliorates OBX-induced depressive-like behaviors by increasing neurogenesis in the DG through activation of the Akt/GSK-3ß/ß-catenin pathway.