Cuprizone Affects Hypothermia-Induced Neuroprotection and Enhanced Neuroblast Differentiation in the Gerbil Hippocampus after Ischemia.

In the present study, we investigated the effects of cuprizone on cell death, glial activation, and neuronal plasticity induced by hypothermia after ischemia in gerbils. Food was supplemented with cuprizone at 0.2% ad libitum for eight weeks. At six weeks after diet feeing, gerbils received transient forebrain ischemia with or without hypothermic preconditioning. Cuprizone treatment for 8 weeks increased the number of astrocytes, microglia, and pro-inflammatory cytokine levels in the hippocampus. In addition, cuprizone treatment significantly decreased the number of proliferating cells and neuroblasts in the dentate gyrus. Brain ischemia caused cell death, disruption of myelin basic proteins, and reactive gliosis in CA1. In addition, ischemia significantly increased pro-inflammatory cytokines and the number of proliferating cells and differentiating neuroblasts in the dentate gyrus. In contrast, hypothermic conditioning attenuated these changes in CA1 and the dentate gyrus. However, cuprizone treatment decreased cell survival induced by hypothermic preconditioning after ischemia and increased the number of reactive microglia and astrocytes in CA1 as well as that of macrophages in the subcallosal zone. These changes occurred because the protective effect of hypothermia in ischemic damage was disrupted by cuprizone administration. Furthermore, cuprizone decreased ischemia-induced proliferating cells and neuroblasts in the dentate gyrus.

Differential roles of exogenous protein disulfide isomerase A3 on proliferating cell and neuroblast numbers in the normal and ischemic gerbils.

INTRODUCTION: We examined the effects of exogenous protein disulfide isomerase A3 (PDIA3) on hippocampal neurogenesis in gerbils under control and ischemic damage. METHODS: To facilitate the delivery of PDIA3 to the brain, we constructed Tat-PDIA3 protein and administered vehicle (10% glycerol) or Tat-PDIA3 protein once a day for 28 days. On day 24 of vehicle or Tat-PDIA3 treatment, ischemia was transiently induced by occlusion of both common carotid arteries for 5 min. RESULTS: Administration of Tat-PDIA3 significantly reduced ischemia-induced spontaneous motor activity, and the number of NeuN-positive nuclei in the Tat-PDIA3-treated ischemic group was significantly increased in the CA1 region compared to that in the vehicle-treated ischemic group. Ki67- and DCX-immunoreactive cells were significantly higher in the Tat-PDIA3-treated group compared to the vehicle-treated control group. In vehicle- and Tat-PDIA3-treated ischemic groups, the number of Ki67- and DCX-immunoreactive cells was significantly higher as compared to those in the vehicle- and Tat-PDIA3-treated control groups, respectively. In the dentate gyrus, the numbers of Ki67-immunoreactive cells were comparable between vehicle- and Tat-PDIA3-treated ischemic groups, while more DCX-immunoreactive cells were observed in the Tat-PDIA3-treated group. Transient forebrain ischemia increased the expression of phosphorylated cAMP-response element-binding protein (pCREB) in the dentate gyrus, but the administration of Tat-PDIA3 robustly increased pCREB-positive nuclei in the normal gerbils, but not in the ischemic gerbils. Brain-derived neurotrophic factor (BDNF) mRNA expression was significantly increased in the Tat-PDIA3-treated group compared to that in the vehicle-treated group. Transient forebrain ischemic increased BDNF mRNA levels in both vehicle- and Tat-PDIA3-treated groups, and there were no significant differences between groups. CONCLUSIONS: These results suggest that Tat-PDIA3 enhances cell proliferation and neuroblast numbers in the dentate gyrus in normal, but not in ischemic gerbils, by increasing BDNF mRNA and phosphorylation of pCREB.

Role of pyridoxine in GABA synthesis and degradation in the hippocampus.

Pyridoxal-5'-phosphate, the active form of vitamin B6, is associated with activities of several enzymes and the treatment of various neurological disorders. Here, we investigated the effects of pyridoxine on the immunoreactivity and protein levels of γ-aminobutyric acid (GABA)-synthesizing and degradation enzymes such as glutamic acid decarboxylase (GAD), GABA transaminase (GABA-T), and succinic semialdehyde dehydrogenase (SSADH), in the hippocampus of mice. The mice intraperitonially received physiological saline and 350 mg/kg pyridoxine, twice a day for 21 days, and were euthanized 2 h after the final dose. In the vehicle-treated group, we observed GAD67 immunoreactivity in the stratum pyramidale of the CA1 and CA3 region, Schaffer collateral, polymorphic layer, and outer granule cell layer of the dentate gyrus. Pyridoxine administration significantly increased GAD67 immunoreactivity, while significantly decreasing GABA-T immunoreactivity in pyridoxine-treated mouse hippocampi (CA1 region and dentate gyrus). In the stratum lacunosum-moleculare of CA1 region, GABA-T immunoreactivity was significantly increased in the pyridoxine-treated group compared to that in the vehicle-treated group, although GAD67 immunoreactivity was similarly observed in these groups. Alternatively, there were no significant differences in SSADH immunoreactivity in any regions of the hippocampus between the vehicle- and pyridoxine-treated groups. Western blot analysis showed significant increases in GAD67 and GABA-T protein levels in the pyridoxine-treated group compared with those in the vehicle-treated group. Therefore, pyridoxine administration facilitates GABA turnover in mouse hippocampus by modulating the GABA-synthesizing and degradation enzymes.

Melatonin ameliorates cuprizone-induced reduction of hippocampal neurogenesis, brain-derived neurotrophic factor, and phosphorylation of cyclic AMP response element-binding protein in the mouse dentate gyrus.

INTRODUCTION: The aim of this study was to investigate the effects of cuprizone on adult hippocampal neurogenesis in naïve mice. Additionally, we also studied how melatonin affects the neuronal degeneration induced by cuprizone. METHODS: Eight-week-old male C57BL/6J mice were randomly divided into three groups: (a) the control group, (b) the group treated with cuprizone only, and (c) the group treated with both cuprizone and melatonin. Cuprizone was administered with food at 0.2% ad libitum for 6 weeks. Melatonin was also administered with tap water at 6 g/L ad libitum for 6 weeks; the animals were then euthanized for immunohistochemistry with Ki67, doublecortin (DCX), glucose transporter 3 (GLUT3), and phosphorylation of cyclic adenosine monophosphate (AMP) response element binding (pCREB); double immunofluorescence of neuronal nuclei (NeuN) and myelin basic protein (MBP); and Western blot analysis of brain-derived neurotrophic factor (BDNF) expression to reveal the effects of cuprizone and melatonin on cell damage and hippocampal neurogenesis. RESULTS: Administration of cuprizone significantly decreased the number of differentiating (DCX-positive) neuroblasts and proliferating (Ki67-positive) cells in the dentate gyrus. Moreover, cuprizone administration decreased glucose utilization (GLUT3-positive cells) and cell transcription (pCREB-positive cells and BDNF protein expression) in the dentate gyrus. Administration of melatonin ameliorated the cuprizone-induced reduction of differentiating neuroblasts and proliferating cells, glucose utilization, and cell transcription. CONCLUSION: The results of the study suggest that cuprizone treatment disrupts hippocampal neurogenesis in the dentate gyrus by reducing BDNF levels and decreasing the phosphorylation of CREB. These effects were ameliorated by melatonin treatment.

Leaf extracts from Dendropanax morbifera Léveille mitigate mercury-induced reduction of spatial memory, as well as cell proliferation, and neuroblast differentiation in rat dentate gyrus.

BACKGROUND: The brain is susceptible to methylmercury toxicity, which causes irreversible damage to neurons and glia and the leaf extract Dendropanax morbifera Léveille (DML) has various biological functions in the nervous system. In this study, we examined the effects of DML on mercury-induced proliferating cells and differentiated neuroblasts. METHODS: Dimethylmercury (5 µg/kg) and galantamine (5 mg/kg) was administered intraperitoneally and/or DML (100 mg/kg) was orally to 7-week-old rats every day for 36 days. One hour after the treatment, novel object recognition test was examined. In addition, spatial probe tests were conducted on the 6th day after 5 days of continuous training in the Morris swim maze. Thereafter, the rats were euthanized for immunohistochemical staining analysis with Ki67 and doublecortin and measurement for acetylcholinesterase (AChE) activity. RESULTS: Dimethylmercury-treated rats showed reduced discrimination index in novel object recognition test and took longer to find the platform than did control group. Compared with dimethylmercury treatment alone, supplementation with DML or galatamine significantly ameliorated the reduction of discrimination index and reduced the time spent to find the platform. In addition, the number of platform crossings was lower in the dimethylmercury-treated group than in controls, while the administration of DML or galantamine significantly increased the number of crossings than did dimethylmercury treatment alone. Proliferating cells and differentiated neuroblasts, assessed by Ki67 and doublecortin immunohistochemical staining was significantly decreased in the dimethylmercury treated group versus controls. Supplementation with DML or galantamine significantly increased the number of proliferating cells and differentiated neuroblasts in the dentate gyrus. In addition, treatment with dimethylmercury significantly increased AChE activity in hippocampal homogenates, while treatment with dimethylmercury+DML or dimethylmercury+galantamine significantly ameliorated this increase. CONCLUSIONS: These results suggest that DML may be a functional food that improves dimethylmercury-induced memory impairment and ameliorates dimethylmercury-induced reduction in proliferating cells and differentiated neuroblasts, and demonstrates corresponding activation of AChE activity in the dentate gyrus.

Methionine-Choline Deprivation Impairs Adult Hippocampal Neurogenesis in C57BL/6 Mice.

Methionine and choline, which are essential nutrients for mammalian animals, are important for cell composition, as metabolic factors, and for the synthesis of other biochemical compounds for cell metabolism. Methionine and choline, which are methyl group donors, play key roles in the homocysteine cycle and neuronal development and maintenance. In this study, we investigated the effects of methionine and choline deficiency on adult hippocampal neurogenesis and neural stem cell (NSC) lineage in the adult stage. For this study, we divided C57BL/6 mice into three groups as follows: normal chow (NC)-fed, methionine choline sufficient (MCS) diet-fed, and methionine choline deficient (MCD) diet-fed mice. The mice were fed the NC, MCS, and MCD diets for 4 weeks from the age of 8 weeks. MCD diet-fed mice showed significantly decreased proliferation and differentiation of NSCs when compared with the NC diet-fed or MCS diet-fed mice. In addition, the survival of newly generated neurons was critically impaired in the MCD diet-fed mice. We confirmed a decrease in the proliferation and differentiation of NSCs after 4 weeks of MCD diet administration, compared with that in NC- and MCS diet-fed mice. MCD diet critically impaired NSCs survival and survival of neurons during the 4 weeks. The number of phosphorylated cyclic AMP response element binding (pCREB) protein immunoreactive nuclei was decreased in the MCD diet-fed mice compared with that in the NC- or MCS diet-fed group. These results suggest that suitable levels of methionine and choline are essential for the maintenance of hippocampal neurogenesis in mice and affect NSC proliferation and differentiation through phosphorylation of CREB.

Effects of long-term exposure to aluminum in the hippocampus in the type 2 diabetes model rats.

We investigated the long-term effects of aluminum (Al) exposure in the hippocampus in Zucker diabetic fatty (ZDF) rats and Zucker lean control (ZLC) rats. Six-week-old ZLC and ZDF rats were randomly divided into Al- and non-Al-groups. They were sacrificed 27 weeks after Al exposure (2000 ppm) through drinking water. Al exposure did not affect physiological parameters such as the body weight and blood glucose levels, but the prolonged diabetic condition had significant effects on the body weight and blood glucose levels. To determine the effects of diabetes and Al exposure on the neural plasticity and inflammatory response in the hippocampus, we examined the levels of doublecortin (DCX), N-methyl-d-aspartate receptors (NMDAR1, NMDAR2A, and NMDAR2B), and ionized calcium-binding adapter molecule 1 (Iba-1) in the hippocampus. DCX immunohistochemical staining revealed that Al exposure significantly reduced neuronal differentiation in both ZLC and ZDF rats. In particular, ZDF rats showed significantly decreased DCX immunoreactive neuroblasts compared with ZLC rats after aluminum exposure. In contrast, the expression of postsynaptic NMDARs was altered only in ZDF-Al rats; the protein expression level of NMDAR1 was reduced, but that of NMDAR2B increased in the hippocampus. Iba-1-immunoreactive microglia with morphological changes, including increased cytoplasm and retracted processes, were detected in the long-term diabetic condition and in the case of the co-existence of diabetes and Al exposure. Al exposure aggravated the diabetes-induced reduction of neuroblast differentiation and NMDAR signaling and facilitated the morphological changes associated with inflammatory activation in microglia in the hippocampus. However, further studies are still needed to confirm these findings.

Adult Hippocampal Neurogenesis Can Be Enhanced by Cold Challenge Independently From Beigeing Effects.

In this study, we investigated the effects of cold challenge on adult hippocampal neurogenesis (AHN) and hippocampal gene expression and whether these are mediated by beigeing of peripheral fat tissues. Cold challenge (6 ± 2°C) for 1 and 4 weeks was found to induce beigeing effects in inguinal white adipose tissue based on hematoxylin and eosin staining as well as uncoupled protein-1 immunohistochemical staining. In the hippocampus, cold challenge for 1 or 4 weeks increased dentate gyrus neurogenesis and expression of genes related to AHN, including notch signaling, G protein-coupled receptor signaling, and adrenergic beta receptor-1. However, this enhancement of neurogenesis and gene expression by cold challenge was not shown by administration of CL 316,243, which induces peripheral beigeing similar to cold challenge but does not cross the blood-brain barrier. These results suggest that cold challenge promotes AHN and central expression of AHN-related, signaling, and ß1-adrenergic receptors genes, and that peripheral beigeing by itself is not sufficient to mediate these effects. Considering the increase in AHN and gene expression changes, cold challenge may offer a novel approach to hippocampal modulation.

Postnatal changes in constitutive cyclooxygenase­2 expression in the mice hippocampus and its function in synaptic plasticity.

Although the expression of cyclooxygenase-2 (COX-2) is closely associated with inflammation in the brain, it is constitutively expressed in the brain, and its expression is regulated by synaptic activity. The present study investigated postnatal expression of COX­2 in the hippocampus in C57BL/6 mice at postnatal days (P) 1, 7, 14, 28, and 56. In addition, the presented study examined the effects of COX­2 on synaptic plasticity through Arc, phosphorylated cAMP response element­binding protein (pCREB), N­methyl­d­aspartate receptor 1 (GluN1), and GluN2A/2B immunohistochemistry, which was performed on COX­2 knockout (KO) and wild­type (WT) mice. Extremely weak COX­2 immunoreactivity was detected in the hippocampal CA1­3 areas in addition to the dentate gyrus at P1. Conversely, COX­2 immunoreactivity was observed in the stratum pyramidale of the CA1­3 regions and in the outer granule cell layer of the dentate gyrus at P7. Additionally, although peak COX­2 immunoreactivity was observed in all hippocampal sub­regions, including the dentate gyrus at P14, it was significantly decreased at P14. Finally, COX­2 immunoreactivity and the distribution pattern seen at P56 in the hippocampal CA1­3 regions were similar to those observed at P28, whereas, they were identified in the inner half of the granule cell layer of the dentate gyrus. The western blot analysis revealed that the COX­2 protein levels peaked at P14 and were decreased at P28 and P56. Additionally, the number of Arc and pCREB immunoreactive cells as well as GluN1 and GluN2A/2B immunoreactivity of COX­2 KO mice were significantly decreased in the dentate gyrus when compared with that in WT mice. Taken together, the results of the present study suggest that COX­2 serves an important role in synaptic plasticity in the dentate gyrus and changes in the levels of its constitutive expression are associated with the hippocampal dentate gyrus postnatal development.

Protein disulfide-isomerase A3 significantly reduces ischemia-induced damage by reducing oxidative and endoplasmic reticulum stress.

Ischemia causes oxidative stress in the endoplasmic reticulum (ER), accelerates the accumulation of unfolded and misfolded proteins, and may ultimately lead to neuronal cell apoptosis. In the present study, we investigated the effects of protein disulfide-isomerase A3 (PDIA3), an ER-resident chaperone that catalyzes disulfide-bond formation in a subset of glycoproteins, against oxidative damage in the hypoxic HT22 cell line and against ischemic damage in the gerbil hippocampus. We also confirmed the neuroprotective effects of PDIA3 by using PDIA3-knockout HAP1 cells. The HT22 and HAP1 cell lines showed effective (dose-dependent and time-dependent) penetration and stable expression of the Tat-PDIA3 fusion protein 24 h after Tat-PDIA3 treatment compared to that in the control-PDIA3-treated group. We observed that the fluorescence for both 2',7'-dichlorofluorescein diacetate (DCF-DA) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), which are markers for the formation of hydrogen peroxide (H2O2)-induced reactive oxygen species and apoptosis, respectively, was higher in HAP1 cells than in HT22 cells. The administration of Tat-PDIA3 significantly reduced the (1) DCF-DA and TUNEL fluorescence in HT22 and HAP1 cells, (2) ischemia-induced hyperactivity that was observed 1 day after ischemia/reperfusion, (3) ischemia-induced neuronal damage and glial (astrocytes and microglia) activation that was observed in the hippocampal CA1 region 4 days after ischemia/reperfusion, and (4) lipid peroxidation and nitric oxide generation in the hippocampal homogenates 3-12 h after ischemia/reperfusion. Transient forebrain ischemia significantly elevated the immunoglobulin-binding protein (BiP) and C/EBP-homologous protein (CHOP) mRNA levels in the hippocampus at 12 h and 4 days after ischemia, relative to those in the time-matched sham-operated group. Administration of Tat-PDIA3 ameliorated the ischemia-induced upregulation of BiP mRNA levels versus the Tat peptide- or control-PDIA3-treated groups, and significantly reduced the induction of CHOP mRNA levels, at 12 h or 4 days after ischemia. Collectively, these results suggest that Tat-PDIA3 acts as a neuroprotective agent against ischemia by attenuating oxidative damage and blocking the apoptotic pathway that is related to the unfolded protein response in the ER.

Phosphoglycerate Mutase 1 Promotes Cell Proliferation and Neuroblast Differentiation in the Dentate Gyrus by Facilitating the Phosphorylation of cAMP Response Element-Binding Protein.

In a previous study, we observed a significant increase in phosphoglycerate mutase 1 (PGAM1) levels after pyridoxine treatment. In the present study, we investigated the effects of PGAM1 on novel object recognition, cell proliferation, and neuroblast differentiation in the dentate gyrus. We generated a Tat-PGAM1 fusion protein to cross the blood-brain barrier and neuronal plasma membrane. We administered the Tat peptide, control-PGAM1, or Tat-PGAM1 fusion protein to 8-week-old mice once a day for 3 weeks and tested novel object recognition memory. The mice were then euthanized to conduct western blot analysis for polyhistidine expression and immunohistochemical analysis for Ki67, doublecortin, and phosphorylated cAMP response element-binding protein. Mice treated with Tat peptide showed similar exploration times for familiar and new objects and the discrimination index was significantly lower in this group than in the control group. Tat-PGAM1 moderately increased the exploration time of new objects when compared to familiar objects, while the discrimination index was significantly higher in the Tat-PGAM1-treated group, but not in the control-PGAM1-treated group, when compared with the control group. Higher PGAM1 protein expression was observed in the hippocampus of Tat-PGAM1-treated mice when compared with the hippocampi of control, Tat peptide-, and control-PGAM1-treated mice, using western blot analysis. In addition, the numbers of proliferating cells and differentiated neuroblasts were significantly lower in the Tat peptide-treated group than in the control group. In contrast, the numbers of proliferating cells and differentiated neuroblasts in the dentate gyrus were higher in the Tat-PGAM1-treated group than in the control group. Administration of Tat-PGAM1 significantly facilitated the phosphorylation of cAMP response element-binding protein in the dentate gyrus. Administration of control-PGAM1 did not show any significant effects on novel object recognition, cell proliferation, and neuroblast differentiation in the dentate gyrus. These results suggest that PGAM1 plays a role in cell proliferation and neuroblast differentiation in the dentate gyrus via the phosphorylation of cAMP response element-binding protein in the hippocampus.

Heat shock protein 70 increases cell proliferation, neuroblast differentiation, and the phosphorylation of CREB in the hippocampus.

In the present study, we investigated the effects of heat shock protein 70 (HSP70) on novel object recognition, cell proliferation, and neuroblast differentiation in the hippocampus. To facilitate penetration into the blood-brain barrier and neuronal plasma membrane, we created a Tat-HSP70 fusion protein. Eight-week-old mice received intraperitoneal injections of vehicle (10% glycerol), control-HSP70, or Tat-HSP70 protein once a day for 21 days. To elucidate the delivery efficiency of HSP70 into the hippocampus, western blot analysis for polyhistidine was conducted. Polyhistidine protein levels were significantly increased in control-HSP70- and Tat-HSP70-treated groups compared to the control or vehicle-treated group. However, polyhistidine protein levels were significantly higher in the Tat-HSP70-treated group compared to that in the control-HSP70-treated group. In addition, immunohistochemical study for HSP70 showed direct evidences for induction of HSP70 immunoreactivity in the control-HSP70- and Tat-HSP70-treated groups. Administration of Tat-HSP70 increased the novel object recognition memory compared to untreated mice or mice treated with the vehicle. In addition, the administration of Tat-HSP70 significantly increased the populations of proliferating cells and differentiated neuroblasts in the dentate gyrus compared to those in the control or vehicle-treated group based on the Ki67 and doublecortin (DCX) immunostaining. Furthermore, the phosphorylation of cAMP response element-binding protein (pCREB) was significantly enhanced in the dentate gyrus of the Tat-HSP70-treated group compared to that in the control or vehicle-treated group. Western blot study also demonstrated the increases of DCX and pCREB protein levels in the Tat-HSP70-treated group compared to that in the control or vehicle-treated group. In contrast, administration of control-HSP70 moderately increased the novel object recognition memory, cell proliferation, and neuroblast differentiation in the dentate gyrus compared to that in the control or vehicle-treated group. These results suggest that Tat-HSP70 promoted hippocampal functions by increasing the pCREB in the hippocampus.

Strain-specific differential expression of astrocytes and microglia in the mouse hippocampus.

Introduction: Genetic background influences neurotransmitter expression and function of the hippocampus. Genetic background influences the phenotype of the hippocampus, but expression of neuroglia in hippocampus has not been well established dependent on various mouse strains. Objectives: In this study, we investigated the effects of genetic background on cell population of astrocytes and microglia in eight widely used inbred strains (C57BL/6J, A/J, BALB/c, C3H/HeJ, FVB, 129/SvJ, DBA/1, and DBA/2) and one outbred strain (ICR). Methods: In all mouse strains, glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes and ionized calcium-binding adaptor molecule 1 (Iba-1)-immunoreactive microglia were found in almost all layers of hippocampal CA1-4 regions and the dentate gyrus. Results: We observed significant differences in the number of astrocytes and microglia. In the CA1 and CA3 regions, the number of GFAP-immunoreactive astrocytes was highest in the C3H/HeJ strain, and lowest in the 129/SvJ and FVB strains. In the polymorphic layer of the dentate gyrus, the number was highest in the DBA/1 strain and lowest in the 129/SvJ strain. Among the nine mouse strains, the number of Iba-1-immunoreactive microglia was highest in the CA1 and CA3 regions in the ICR and in the dentate gyrus of the C57BL/6 strain. The CA1 region of the FVB strain and the CA3 region and dentate gyrus of DBA/2 had the lowest number of Iba-1-immunoreactive microglia. Conclusion: These results suggest that the numbers of astrocytes and microglia differ depending on the mouse strain and these differences may be related to strain-dependent function of astrocytes.

Age-dependent changes in vesicular glutamate transporter 1 and 2 expression in the gerbil hippocampus.

Glutamate is a major excitatory neurotransmitter that is stored in vesicles located in the presynaptic terminal. Glutamate is transported into vesicles via the vesicular glutamate transporter (VGLUT). In the present study, the age­associated changes of the major VGLUTs, VGLUT1 and VGLUT2, in the hippocampus were investigated, based on immunohistochemistry and western blot analysis at postnatal month 1 (PM1; adolescent), PM6, PM12 (adult group), PM18 and PM24 (the aged groups). VGLUT1 immunoreactivity was primarily detected in the mossy fibers, Schaffer collaterals and stratum lacunosum­moleculare. By contrast, VGLUT2 immunoreactivity was observed in the granule cell layer and the outer molecular layer of the dentate gyrus, stratum pyramidale, Schaffer collaterals and stratum lacunosum­moleculare in the hippocampal CA1­3 regions. VGLUT1 immunoreactivity and protein levels remained constant across all age groups. However, VGLUT2 immunoreactivity and protein levels decreased in the PM3 group when compared with the PM1 group. VGLUT2 immunoreactivity and protein levels were not altered in the PM12 group; however, they increased in the PM18 group. In addition, in the PM18 group, highly immunoreactive VGLUT2 cells were also identified in the stratum radiatum and oriens of the hippocampal CA1 region. In the PM24 group, VGLUT2 immunoreactivity and protein levels were significantly decreased and were the lowest levels observed amongst the different groups. These results suggested that VGLUT1 may be less susceptible to the aging process; however, the increase of VGLUT2 in the non­pyramidal cells in the PM18 group, and the consequent decrease in VGLUT2, may be closely linked to age­associated memory impairment in the hippocampus.

Hypothyroidism increases cyclooxygenase-2 levels and pro-inflammatory response and decreases cell proliferation and neuroblast differentiation in the hippocampus.

The present study investigated the effects of hypothyroidism on cyclooxygenase-2 (COX-2) and pro­inflammatory cytokines in the dentate gyrus to elucidate the roles of COX­2 in the hypothyroid hippocampus. Hypothyroidism was induced in rats by treating with 0.03% 2­mercapto­1­methyl­imidazole dissolved in drinking water for 5 weeks. The animals were sacrificed at 12 weeks of age. Hypothyroidism rats exhibited decreased triiodothyronine and thyroxine levels in the serum, while the levels of thyroid­stimulating hormone and the weight of thyroid glands were significantly higher in the hypothyroid rats compared with those in the vehicle­treated group. COX­2 immunoreactivity was significantly increased in the hippocampal CA2/3 region and the dentate gyrus compared with the vehicle­treated group. Levels of pro­inflammatory cytokines including interleukin (IL)­1ß, IL­6 and tumor necrosis factor­α were significantly higher in the hippocampal homogenates of hypothyroid rats. Cell proliferation and neuroblast differentiation based on Ki67 and doublecortin immunohistochemistry were decreased in the dentate gyrus of hypothyroid rats compared with those in the vehicle­treated group. These results suggested that hypothyroidism­mediated COX­2 expression affected hippocampal plasticity by upregulating the levels of pro­inflammatory cytokines in the hippocampus. Therefore, COX­2 may be suggested as a candidate molecule for preventing hypothyroidism­induced neurological side effects.

Changes of myelin basic protein in the hippocampus of an animal model of type 2 diabetes.

In this study, we observed chronological changes in the immunoreactivity and expression level of myelin basic protein (MBP), one of the most abundant proteins in the central nervous system, in the hippocampus of Zucker diabetic fatty (ZDF) rats and their control littermates (Zucker lean control; ZLC). In the ZLC group, body weight steadily increased with age; the body weight of the ZDF group, however, peaked at 30 weeks of age, and subsequently decreased. Based on the changes of body weight, animals were divided into the following six groups: early (12-week), middle (30-week), and chronic (52-week) diabetic groups and their controls. MBP immunoreactivity was found in the alveus, strata pyramidale, and lacunosum-moleculare of the CA1 region, strata pyramidale and radiatum of the CA3 region, and subgranular zone, polymorphic layer, and molecular layer of the dentate gyrus. MBP immunoreactivity was lowest in the hippocampus of 12-week-old rats in the ZLC group, and highest in 12-week-old rats in the ZDF group. Diabetes increased MBP levels in the 12-week-old group, while MBP immunoreactivity decreased in the 30-week-old group. In the 52-week-old ZLC and ZDF groups, MBP immunoreactivity was detected in the hippocampus, similar to the 30-week-old ZDF group. Western blot results corroborated with immunohistochemical results. These results suggested that changes in the immunoreactivity and expression of MBP in the hippocampus might be a compensatory response to aging, while the sustained levels of MBP in diabetic animals could be attributed to a loss of compensatory responses in oligodendrocytes.

Bacopa monnieri extract improves novel object recognition, cell proliferation, neuroblast differentiation, brain-derived neurotrophic factor, and phosphorylation of cAMP response element-binding protein in the dentate gyrus.

Bacopa monnieri is a medicinal plant with a long history of use in Ayurveda, especially in the treatment of poor memory and cognitive deficits. In the present study, we hypothesized that Bacopa monnieri extract (BME) can improve memory via increased cell proliferation and neuroblast differentiation in the dentate gyrus. BME was administered to 7-week-old mice once a day for 4 weeks and a novel object recognition memory test was performed. Thereafter, the mice were euthanized followed by immunohistochemistry analysis for Ki67, doublecortin (DCX), and phosphorylated cAMP response element-binding protein (CREB), and western blot analysis of brain-derived neurotrophic factor (BDNF). BME-treated mice showed moderate increases in the exploration of new objects when compared with that of familiar objects, leading to a significant higher discrimination index compared with vehicle-treated mice. Ki67 and DCX immunohistochemistry showed a facilitation of cell proliferation and neuroblast differentiation following the administration of BME in the dentate gyrus. In addition, administration of BME significantly elevated the BDNF protein expression in the hippocampal dentate gyrus, and increased CREB phosphorylation in the dentate gyrus. These data suggest that BME improves novel object recognition by increasing the cell proliferation and neuroblast differentiation in the dentate gyrus, and this may be closely related to elevated levels of BDNF and CREB phosphorylation in the dentate gyrus.

Pyridoxine improves hippocampal cognitive function via increases of serotonin turnover and tyrosine hydroxylase, and its association with CB1 cannabinoid receptor-interacting protein and the CB1 cannabinoid receptor pathway.

BACKGROUND: In the present study, we investigated the effects of pyridoxine on hippocampal functions and changes in protein profiles based on the proteomic approach. METHODS: Eight-week-old mice received intraperitoneal injections of physiological saline (vehicle) or 350mg/kg pyridoxine twice a day for 21days. RESULTS: Phosphoglycerate mutase 1 was up-regulated, while CB1 cannabinoid receptor-interacting protein 1 (CRIP1) was down-regulated, in the pyridoxine-treated group. Additionally, the serotonin and tyrosine hydroxylase was increased in the hippocampus of the pyridoxine-treated group than in that of the vehicle-treated group. Furthermore, discrimination indices based on the novel object recognition test were significantly higher in the pyridoxine-treated group than in the vehicle-treated group. Administration of CRIP1a siRNA significantly increases the discrimination index as well as cell proliferation and neuroblast differentiation in the dentate gyrus. In addition, the administration of rimonabant, a CB1 cannabinoid receptor antagonist, for 3weeks significantly decreased the novel object recognition memory, the tyrosine hydroxylase level, the amount of cell proliferation, and neuroblast differentiation in the dentate gyrus. Treatment with pyridoxine significantly increased novel object recognition memory, but slightly ameliorated rimonabant-induced reduction in serotonin, the tyrosine hydroxylase level, the amount of cell proliferation, and neuroblast differentiation in the dentate gyrus. CONCLUSION: These results suggest that pyridoxine promotes hippocampal functions by increasing serotonin and tyrosine hydroylase immunoreactivity in the hippocampus. This positive effect may be associated with CRIP1a and CB1 cannabinoid receptor function. GENERAL SIGNIFICANCE: Vitamin-B6 enhances hippocampal functions and this is closely associated with CRIP1a and CB1 cannabinoid receptors.

Differential Effects of Low- and High-dose Zinc Supplementation on Synaptic Plasticity and Neurogenesis in the Hippocampus of Control and High-fat Diet-fed Mice.

In the present study, we investigated the concentration-dependent effect of zinc (Zn) supplementation on the adult hippocampus in a high-fat diet (HFD)-fed obese mouse model. Four-weeks after HFD- and control diet (CD)-feeding, mice were provided with low (15 ppm) or high (60 ppm) doses of Zn in their drinking water for additional 4 more weeks along with their respective diets. Compared to the CD-fed mice, HFD-feeding elicited the reduction of neurogenic markers such as nestin, Ki67, doublecortin (DCX), and 5-bromo-2'-deoxyuridine (BrdU) in the dentate gyrus. Additionally, HFD-feeding reduced the levels of synaptic markers (synaptophysin and N-methyl-D-aspartate receptor) and brain-derived neurotrophic factor (BDNF), while lipid peroxidation was significantly increased in the hippocampus of HFD-fed mice. Against detrimental effects of high-dose Zn, low-dose Zn supplementation in CD-fed mice did not yield any remarkable changes in these parameters. Interestingly, administration of low doses of Zn to HFD-induced obese mice prominently ameliorated HFD-induced changes in neurogenic, synaptic plasticity markers and BDNF levels as well as lipid peroxidation in the hippocampus. In contrast, high-dose Zn supplementation in HFD-fed mice exacerbated the reduction of markers for neurogenesis and synaptic plasticity as well as BDNF levels, but not 4-HNE levels, in the hippocampus. These results suggest that low-dose Zn supplementation in obese mice could reverse the HFD-induced reduction in neurogenic and synaptic marker proteins in the hippocampus by reducing lipid peroxidation and improving BDNF expression, while high-dose Zn supplementation exacerbates the reduction of neurogenesis by affecting synaptic markers and BDNF levels in the hippocampus.

Chronic administration of SUMO­1 has negative effects on novel object recognition memory as well as cell proliferation and neuroblast differentiation in the mouse dentate gyrus.

Post­translational modifications have been associated with developmental and aging processes, as well as in the pathogenesis of certain diseases. The present study aimed to investigate the effects of small ubiquitin­like modifier 1 (SUMO­1) on hippocampal dependent memory function, cell proliferation and neuroblast differentiation. To facilitate the delivery of SUMO­1 into hippocampal neurons, a transactivator of transcription (Tat)­SUMO­1 fusion protein was constructed and mice were divided into two groups: A vehicle (Tat peptide)­treated group and a Tat­SUMO­1­treated group. The vehicle or Tat­SUMO­1 was administered intraperitoneally to 7­week­old mice once daily for 3 weeks, and a novel object recognition test was conducted following the final treatment; the animals were sacrificed 2 h following the test for further analysis. Administration of Tat­SUMO­1 significantly decreased exploration of a new object in a novel object recognition test compared with mice in the vehicle­treated group. In addition, cell proliferation and neuroblast differentiation analyses (based on Ki67 and doublecortin immunohistochemistry, respectively) revealed that the administration of Tat­SUMO­1 significantly reduced cell proliferation and neuroblast differentiation in the dentate gyrus. These results suggested that chronic supplementation of Tat­SUMO­1 affects hippocampal functions by decreasing cell proliferation and neuroblast differentiation in the mouse dentate gyrus.