Effects of Scopolamine and Melatonin Cotreatment on Cognition,Neuronal Damage,and Neurogenesis in the Mouse Dentate Gyrus

It has been demonstrated that melatonin plays important roles in memory improvement and promotes neurogenesis in experimental animals. We examined effects of melatonin on cognitive deficits, neuronal damage, cell proliferation, neuroblast differentiation and neuronal maturation in the mouse dentate gyrus after cotreatment of scopolamine (anticholinergic agent) and melatonin. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were intraperitoneally injected for 2 and/or 4 weeks to 8-week-old mice. Scopolamine treatment induced significant cognitive deficits 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly improved spatial learning and short-term memory impairments. Two and 4 weeks after scopolamine treatment, neurons were not damaged/dead in the dentate gyrus, in addition, no neuronal damage/death was shown after cotreatment of scopolamine and melatonin. Ki67 (a marker for cell proliferation)- and doublecortin (a marker for neuroblast differentiation)-positive cells were significantly decreased in the dentate gyrus 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly increased Ki67- and doublecortin-positive cells compared with scopolamine-treated group. However, double immunofluorescence for NeuN/BrdU, which indicates newly-generated mature neurons, did not show double-labeled cells (adult neurogenesis) in the dentate gyrus 2 and 4 weeks after cotreatment of scopolamine and melatonin. Our results suggest that melatonin treatment recovers scopolamine-induced spatial learning and short-term memory impairments and restores or increases scopolamine-induced decrease of cell proliferation and neuroblast differentiation, but does not lead to adult neurogenesis (maturation of neurons) in the mouse dentate gyrus following scopolamine treatment.     

PEP-1-frataxin significantly increases cell proliferation and neuroblast differentiation by reducing lipid peroxidation in the mouse dentate gyrus

Frataxin plays important roles in the mitochondrial respiratory chain and in the differentiation of neurons during early development. In this study, we observed the effects of frataxin on cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus. For this, we constructed an expression vector, PEP-1, that was fused with frataxin to create a PEP-1-frataxin fusion protein that easily penetrated frataxin into the blood-brain barrier. Three mg/kg PEP-1-frataxin was intraperitoneally administered to mice once a day for 2 weeks. The administration of PEP-1 alone did not result in any significant changes in the number of Ki67-positive cells and doublecortin (DCX)-immunoreactive neuroblasts in the mouse dentate gyrus. However, the administration of PEP-1-frataxin significantly increased the number of Ki67-positive cells and DCX-immunoreactive neuroblasts in the mouse dentate gyrus. In addition, PEP-1-frataxin significantly reduced 4-hydroxynonenal protein levels and malondialdehyde formation, while Cu, Zn-superoxide dismutase protein levels were maintained. These results suggest that frataxin effectively increased cell proliferation and neuroblast differentiation by decreasing lipid peroxidation in the dentate gyrus.     

Metformin Normalizes Type 2 Diabetes-Induced Decrease in Cell Proliferation and Neuroblast Differentiation in the Rat Dentate Gyrus

In this study, we observed the effects of metformin, one of the most widely prescribed drugs for the treatment of type 2 diabetes, on cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus (SZDG) in Zucker diabetic fatty (ZDF) rats, which are a model for type 2 diabetes. For this, metformin was administered orally once a day to 14-week-old ZDF rats for 2 weeks and the animals were sacrificed at 16 weeks of age. During this period, blood glucose levels were higher in the vehicle-treated ZDF rats than in the Zucker lean control (ZLC) rats. Metformin treatment significantly decreased the blood glucose levels from 15.5 weeks of age. In the SZDG, Ki67 (a marker for cell proliferation)- and doublecortin (DCX, a marker for differentiated neuroblasts)-immunoreactive cells were much lower in the vehicle-treated ZDF rats than in the ZLC rats. In the metformin-treated ZDF group, Ki67- and DCX-immunoreactive cells were significantly increased in the SZDG compared to those in the vehicle-treated ZDF group. These results suggest that diabetes significantly reduces cell proliferation and neuroblast differentiation in the SZDG and that metformin treatment normalizes the reduction of cell proliferation and neuroblast differentiation in the SZDG in diabetic rats.     

Pyridoxine Enhances Cell Proliferation and Neuroblast Differentiation by Upregulating the GABAergic System in the Mouse Dentate Gyrus

We investigated the effects of pyridoxine (vitamin B₆) on cell death, cell proliferation, neuroblast differentiation, and the GABAergic system in the mouse dentate gyrus. We administered pyridoxine (350 mg/kg intraperitoneally) to 8 week old mice twice a day for 14 days and sacrificed them at 10 weeks of age. Pyridoxine treatment did not induce neuronal death or activate microglia in the dentate gyrus, while glial fibrillary acidic protein (GFAP)-positive cells were significantly increased in the subgranular zone of the dentate gyrus. The increase in GFAP-positive cells was confirmed to be due to proliferating cells based on double immunofluorescence staining. GFAP-positive cells, which were also labeled with Ki67, a marker for cell proliferation, and doublecortin, a marker for neuroblast differentiation, were significantly increased in the pyridoxine-treated group compared to those in the vehicle-treated group. Pyridoxine treatment also increased the protein levels of glutamic acid decarboxylase (GAD) 67, an enzyme for GABA synthesis, and pyridoxal 5′-phosphate (PNP) oxidase, an enzyme for pyridoxal phosphate synthesis, in the dentate gyrus. These results suggest that pyridoxine treatment distinctly increases cell proliferation, neuroblast differentiation, and upregulated the GABAergic system, as revealed by the increases of GAD67 and PNP oxidase in the mouse dentate gyrus.     

Effects of Adrenalectomy and Replacement Therapy of Corticosterone on Cell Proliferation and Neuroblast Differentiation in the Rat Dentate Gyrus

Newly generated neurons in the dentate gyrus differentiate into mature granule cells. In the present study, we observed the effects of adrenalectomy (ADX) and corticosterone replacement therapy (CRT) on cell death, cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus (SZDG). For this, the animals received vehicle or CRT after ADX, and were sacrificed 5 or 42 days later. Plasma corticosterone levels were very low in the adrenalectomized groups, whereas CRT after ADX significant increased serum corticosterone levels at 42 days, not 5 days, after ADX. ADX induced some neuronal damage in the dentate gyrus at 5 days post-ADX. CRT did not significantly reduce the neuronal damage at 5 days post-ADX; however, neuronal damage was not shown at 42 post-ADX with CRT. Ki67 (a marker for cell proliferation) and doublecortin (DCX, a marker for neuronal differentiation) immunoreaction was detected in the SZDG. ADX transiently increased cell proliferation and neuroblast differentiation 5 days after ADX, not 42 days, after ADX, and the CRT 42 days after ADX prominently decreased cell proliferation and neuroblast differentiation in the dentate gyrus. These results suggest that adrenal corticosteroid hormone is not essential for cell proliferation and neuroblast differentiation in long-term period after ADX.     

Combination Effects of Sodium Butyrate and Pyridoxine Treatment on Cell Proliferation and Neuroblast Differentiation in the Dentate Gyrus of d-Galactose-Induced Aging Model Mice

We previously reported that sodium butyrate (SB), a histone deacetylase inhibitor, robustly increased pyridoxine-induced cell proliferation and neuroblast differentiation in the dentate gyrus of the adult mouse. In this study, we investigated the effects of treatment with SB combined with pyridoxine on cell proliferation and neuroblast differentiation in the dentate gyrus of a mouse model of aging induced by d-galactose (d-gal). d-gal was administered to 20-week-old male mice (d-gal mice) for 10 weeks to induce changes that resemble natural aging in animals. Seven weeks after d-gal (100 mg/kg) treatment, vehicle (physiological saline; d-gal-vehicle mice) and SB (300 mg/kg) combined with pyridoxine (Pyr; 350 mg/kg) were administered to the mice (d-gal-Pyr-SB mice) for 3 weeks. Escape latency under water maze in the d-gal mice was longer than that in the control mice. In the d-gal-Pyr-SB mice, escape latency was similar to that in the control mice. In the d-gal mice, many cells in the granule cell layer of the dentate gyrus showed pyknosis and condensation of the cytoplasm. However, in the d-gal-Pyr-SB mice, such cellular changes were rarely found. Furthermore, the d-gal mice showed a great reduction in cell proliferation (Ki67-positive cells) and neuroblast differentiation (doublecortin-positive neuroblasts) in the dentate gyrus compared to control mice. However, in the d-gal-Pyr-SB mice, cell proliferation and neuroblast differentiation were markedly increased in the dentate gyrus. Furthermore, the administration of pyridoxine with sodium butyrate significantly increased Ser133-phosphorylated cyclic AMP response element binding protein in the dentate gyrus. These results indicate that the combination treatment of Pyr with SB in d-gal mice ameliorated the d-gal-induced reduction in cell proliferation, neuroblast differentiation, and memory deficits.     

Effects of Treadmill Exercise on Cell Proliferation and Differentiation in the Subgranular Zone of the Dentate Gyrus in a Rat Model of Type II Diabetes

In the present study, we investigated the effects of a treadmill exercise on serum glucose levels and Ki67 and doublecortin (DCX) immunoreactivity, which is a marker of cell proliferation expressed during cell cycles except G0 and early G1 and a marker of progenitors differentiating into neurons, respectively, in the subgranular zone of the dentate gyrus (SZDG) using a type II diabetic model. At 6 weeks of age, Zucker lean control (ZLC) and Zucker diabetic fatty (ZDF) rats were put on a treadmill with or without running for 1 h/day/5 consecutive days at 22 m/min for 5 weeks. Body weight was significantly increased in the control (without running)-ZDF rats compared to that in the other groups. In the control groups blood glucose levels were increased by 392.7 mg/dl in the control-ZDF rats and by 143.3 mg/dl in the control-ZLC rats. However, in the exercise groups, blood glucose levels were similar between the exercise-ZLC and ZDF rats: The blood glucose levels were 110.0 and 118.2 mg/dl, respectively. Ki67 positive nuclei were detected in the SZDG in control and exercise groups. The number of Ki67 positive nuclei was significantly high in exercise groups compared to that in the control groups. In addition, Ki67 positive cells were abundant in ZLC groups compared to those in ZDF groups. DCX-immunoreactive structures in the control-ZDF rats were lower than that in the control-ZLC rats. In the exercise groups, DCX-immunoreactive structures (somata and processes with tertiary dendrites) and DCX protein levels were markedly increased in both the exercise-ZLC and ZDF rats compared to that in the control groups. These results suggest that a treadmill exercise reduces blood glucose levels in ZDF rats and increases cell proliferation and differentiation in the SZDG in ZLC and ZDF rats compared to those in control groups.     

Effects of Cu,Zn-Superoxide Dismutase on Cell Proliferation and Neuroblast Differentiation in the Mouse Dentate Gyrus

Oxidative stress is one of the most important factors in reducing adult hippocampal neurogenesis in the adult brain. In this study, we observed the effects of Cu,Zn-superoxide dismutase (SOD1) on lipid peroxidation, cell proliferation, and neuroblast differentiation in the mouse dentate gyrus using malondialdehyde (MDA), Ki67, and doublecortin (DCX), respectively. We constructed an expression vector, PEP-1, fused PEP-1 with SOD1, and generated PEP-1-SOD1 fusion protein. We administered PEP-1 and 100 or 500 μg PEP-1-SOD1 intraperitoneally once a day for 3 weeks and sacrificed at 30 min after the last administrations. PEP-1 administration did not change the MDA levels compared to those in the vehicle-treated group, while PEP-1-SOD1 treatment significantly reduced MDA levels compared to the vehicle-treated group. In the PEP-1-treated group, the number of Ki67-positive nuclei was similar to that in the vehicle-treated group. In the 100 μg PEP-1-SOD1-treated group, the number of Ki67-positive nuclei was slightly decreased; however, in the 500 μg PEP-1-SOD1-treated group, Ki67-positive nuclei were decreased to 78.5% of the vehicle-treated group. The number of DCX-positive neuroblasts in the PEP-1-treated group was similar to that in the vehicle-treated group. However, the arborization of DCX-positive neuroblasts was significantly decreased in both the 100 and 500 μg PEP-1-SOD1-treated groups compared to that in the vehicle-treated group. The number of DCX-positive neuroblasts with tertiary dendrites was markedly decreased in the 500 μg PEP-1-SOD1-treated group. These results suggest that a SOD1 supplement to healthy mice may not be necessary to modulate cell proliferation and neuroblast differentiation in the dentate gyrus.     

Reduced cell proliferation and neuroblast differentiation in the dentate gyrus of high fat diet-fed mice are ameliorated by metformin and glimepiride treatment

We investigated the effects of a high-fat diet (HFD) and the subsequent treatment of metformin (met) and glimepiride (glim), which are widely prescribed for type 2 diabetes, on cell proliferation and neuroblast differentiation using Ki67 and doublecortin (DCX) immunohistochemistry, respectively. Animals were fed low-fat diet (LFD) or HFD for 8 weeks. After 5 weeks of the HFD treatment, met alone or met + glim was administered orally once a day for 3 weeks. Body weight and food intake were much higher in the HFD + vehicle-treated group than the LFD-treated group. The administration of met or met + glim to the HFD-treated group resulted in a decrease in weight gain and food intake. Ki67-immunoreactive (⁺) nuclei, DCX⁺ neuroblasts and brain-derived neurotrophic factor (BDNF) protein levels were markedly decreased in the dentate gyrus (DG) of the HFD + vehicle-treated group compared to the LFD-treated group. The administration of met or met + glim to the HFD-treated group prevented the reduction of Ki67⁺ nuclei, DCX⁺ neuroblasts, BDNF levels in the DG. The intraventricular injection of K252a (a BDNF receptor blocker) to the HFD-treated group treated met or met + glim distinctively lowered the reduction of cell proliferation and neuroblast differentiation induced by HFD. These results suggest that a HFD significantly reduces cell proliferation and neuroblast differentiation by reducing BDNF levels and these effects are ameliorated by treatment with met or met + glim.     

Synergistic Effects of Sodium Butyrate,a Histone Deacetylase Inhibitor,on Increase of Neurogenesis Induced by Pyridoxine and Increase of Neural Proliferation in the Mouse Dentate Gyrus

We previously observed that pyridoxine (vitamin B₆) significantly increased cell proliferation and neuroblast differentiation without any neuronal damage in the hippocampus. In this study, we investigated the effects of sodium butyrate, a histone deacetylase (HDAC) inhibitor which serves as an epigenetic regulator of gene expression, on pyridoxine-induced neural proliferation and neurogenesis induced by the increase of neural proliferation in the mouse dentate gyrus. Sodium butyrate (300 mg/kg, subcutaneously), pyridoxine (350 mg/kg, intraperitoneally), or combination with sodium butyrate were administered to 8-week-old mice twice a day and once a day, respectively, for 14 days. The administration of sodium butyrate significantly increased acetyl-histone H3 levels in the dentate gyrus. Sodium butyrate alone did not show the significant increase of cell proliferation in the dentate gyrus. But, pyridoxine alone significantly increased cell proliferation. Sodium butyrate in combination with pyridoxine robustly enhanced cell proliferation and neurogenesis induced by the increase of neural proliferation in the dentate gyrus, showing that sodium butyrate treatment distinctively enhanced development of neuroblast dendrites. These results indicate that an inhibition of HDAC synergistically promotes neurogenesis induced by a pyridoxine and increase of neural proliferation.     

Effects of Sensitive to Apoptosis Gene Protein on Cell Proliferation, Neuroblast Differentiation, and Oxidative Stress in the Mouse Dentate Gyrus

Sensitive to apoptosis gene (SAG) protein is a redox-inducible protein that protects cells against apoptosis induced by redox agents. In this study, we observed effects of SAG on cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus (DG) using Ki67 and doublecortin (DCX), respectively. For easy penetration into neurons, Tat-SAG expression vector was constructed by ligation with SAG and expression vector, Tat, in-frame with six histidine open-reading frames to generate the expression vector, and cloned into E. coli DH5α cells. One or 5?mg/kg Tat-SAG fusion protein (Tat-SAG) was intraperitoneally administered to mice once a day for 3?weeks. The administration of Tat-SAG significantly increased the number of 5-bromodeoxyuridine positive cells, Ki67 positive cells and DCX immunoreactive neuroblast in the mouse DG: Especially, in the 5?mg/kg Tat-SAG-treated mice, DCX positive neuroblasts showed a well-developed arborization of tertiary dendrites in the DG. On the other hand, we examined that the administration of Tat-SAG significantly reduced the DNA damage and lipid peroxidation judging from 8-hydroxy-2'-deoxyguanosine and 4-hydroxynonenal immunohistochemistry: The decrease was much more distinct in the 5?mg/kg Tat-SAG-treated mice than 1?mg/kg Tat-SAG-treated mice. This result suggests that SAG significantly increases cell proliferation, neuroblast differentiation and oxidative stress in normal states.     

Additive or Synergistic Effects of Aluminum on the Reduction of Neural Stem Cells, Cell Proliferation, and Neuroblast Differentiation in the Dentate Gyrus of High-Fat Diet-Fed Mice

Aluminum is the most plentiful metal on the Earth’s crust, and its usage in cooking utensils, cosmetics, drinking containers, food additives, pharmaceutical products, and building materials provides many opportunities for potential aluminum consumption. However, its toxicity is low and harmful effects only develop with large-scale deposition of aluminum. In this study, we investigated the effects of subchronic exposure to aluminum (40 mg/kg/day) on neural stem cells, cell proliferation, neuroblast differentiation, and mature neurons in the dentate gyrus of the hippocampus. These experiments were performed in both high-fat diet and low-fat diet-fed C57BL/6J mice via immunohistochemistry using the relevant marker for each cell type, including nestin, Ki67, doublecortin, and NeuN. Subchronic exposure to aluminum in both low-fat and high-fat diet-fed mice reduced neural stem cells, cell proliferation, and neuroblast differentiation without any changes in mature neurons. Furthermore, this reduction effect was exacerbated in high-fat diet-fed mice. These results suggest that aluminum accelerates the reduction of neural stem cells, cell proliferation, and neuroblast differentiation additively or synergistically in high-fat diet-fed mice without any harmful changes in mature neurons.     

Effects of <Emphasis Type="Italic">Melissa officinalis</Emphasis> L. (Lemon Balm) Extract on Neurogenesis Associated with Serum Corticosterone and GABA in the Mouse Dentate Gyrus

Lemon balm, leaves of Melissa officinalis L., has been used for anti-anxiety and spasmolytics. We observed the extract of Melissa officinalis L. (MOE) on cell proliferation and neuroblast differentiation in the hippocampal dentate gyrus (DG) of middle-aged mice (12 months of age) using Ki67 and doublecortin (DCX), respectively. We also observed changes in corticosterone, GAD67 and GABA-transaminase (GABA-T) to check their possible mechanisms related to neurogenesis. We administered 50 or 200 mg/kg MOE to the animals once a day for 3 weeks. For labeling of newly generated cells, we also administered 5-bromodeoxyuridine (BrdU) twice a day for 3 days from the day of the first MOE treatment. Administration of 50 or 200 mg/kg MOE dose-dependently increased Ki67 positive nuclei to 244.1 and 763.9% of the vehicle-treated group, respectively. In addition, 50 or 200 mg/kg MOE significantly increased DCX positive neuroblasts with well-developed (tertiary) dendrites. Furthermore, MOE administration significantly increased BrdU/calbindin D-28 k double labeled cells (integrated neurons into granule cells in the DG) to 245.2% of the vehicle-treated group. On the other hand, administration of MOE reduced corticosterone levels in serum and decreased GABA-T levels in the DG homogenates. These results suggest that MOE increases cell proliferation, neuroblast differentiation and integration into granule cells by decreasing serum corticosterone levels as well as by increasing GABA levels in the mouse DG.     

Cell Proliferation and Neuroblast Differentiation in the Rat Dentate Gyrus After Intrathecal Treatment with Adipose-Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) have emerged as a new therapeutic tool for a number of clinical applications, because they have multipotency and paracrine effects via various factors. In the present study, we investigated the effects of adipose-derived MSC (Ad-MSC) transplantation via intrathecal injection through the cisterna magna on cell proliferation and differentiation of endogenous stem cells in the hippocampal dentate gyrus (DG) using Ki-67 (a marker for proliferating cells), and doublecortin (DCX, a marker for neuroblasts). The transplanted Ad-MSC were detected in the meninges, not in the hippocampal parenchyma. However, the number of Ki-67-immunoreactive cells was significantly increased by 83% in the DG 2 days after single Ad-MSC injection, and by 67% at 23 days after repeated Ad-MSC treatment compared with that in the vehicle-treated group after Ad-MSC transplantation. On the other hand, the number of DCX-immunoreactive cells in the DG was not changed at 2 days after single Ad-MSC injection; however, it was significantly increased by 62% 9 days after single Ad-MSC injection. At 23 days after repeated Ad-MSC application, the number of DCX-immunoreactive cells was much more increased (223% of the vehicle-treated group). At this time point, DCX protein levels were also significantly increased compared with those in the vehicle-treated group. These results suggest that the intrathecal injection of Ad-MSC could enhance endogenous cell proliferation, and the repeated Ad-MSC injection could be more efficient for an enhancement of endogenous cell proliferation and differentiation in the brain.     

Cyclosporine A Reduces Dendritic Outgrowth of Neuroblasts in the Subgranular Zone of the Dentate Gyrus in C57BL/6 Mice

In the present study, we observed the effects of cyclosporine A (CsA), an efficient immunosuppressant, on cell proliferation and neuroblast differentiation in the subgranular zone of the dentate gyrus (SZDG) in normal C57BL/6 mice using Ki67 and doublecortin (DCX) immunohistochemical staining, respectively. At 8 weeks of age, vehicle (physiological saline) or CsA was daily administered (40 mg/kg, i.p.) for 1 week. Animals were sacrificed at 2 weeks after last administration. CsA treatment did not show any influences in neurons, astrocytes and microglia based on immunohistochemistry for its markers, respectively. However, in the CsA-treated group, Fluoro-Jade B, a marker for neurodegeneration, positive cells were found in the SZDG, not in the vehicle-treated group. In the vehicle-treated group, Ki67 immunoreactive (⁺) nuclei were clustered in the SZDG, whereas in the CsA-treated group Ki67⁺ nuclei were scattered in the SZDG, showing no difference in cell numbers. Numbers of DCX⁺ neuroblasts with well-developed processes (tertiary dendrites) were much lower in the CsA-treated group than those in the vehicle-treated group; however, numbers of DCX⁺ neuroblasts with secondary dendrites were similar in both the groups. These results suggest that CsA significantly reduces dendritic outgrowth and complexity from neuroblasts in the SZDG without any affecting in neurons, astrocytes and microglia in normal mice.     

Galantamine reverses scopolamine-induced behavioral alterations in Dugesia tigrina

In planaria (Dugesia tigrina), scopolamine, a nonselective muscarinic receptor antagonist, induced distinct behaviors of attenuated motility and C-like hyperactivity. Planarian locomotor velocity (pLMV) displayed a dose-dependent negative correlation with scopolamine concentrations from 0.001 to 1.0 mM, and a further increase in scopolamine concentration to 2.25 mM did not further decrease pLMV. Planarian hyperactivity counts was dose-dependently increased following pretreatment with scopolamine concentrations from 0.001 to 0.5 mM and then decreased for scopolamine concentrations ≥1 mM. Planarian learning and memory investigated using classical Pavlovian conditioning experiments demonstrated that scopolamine (1 mM) negatively influenced associative learning indicated by a significant decrease in % positive behaviors from 86 % (control) to 14 % (1 mM scopolamine) and similarly altered memory retention, which is indicated by a decrease in % positive behaviors from 69 % (control) to 27 % (1 mM scopolamine). Galantamine demonstrated a complex behavior in planarian motility experiments since co-application of low concentrations of galantamine (0.001 and 0.01 mM) protected planaria against 1 mM scopolamine-induced motility impairments; however, pLMV was significantly decreased when planaria were tested in the presence of 0.1 mM galantamine alone. Effects of co-treatment of scopolamine and galantamine on memory retention in planaria via classical Pavlovian conditioning experiments showed that galantamine (0.01 mM) partially reversed scopolamine (1 mM)-induced memory deficits in planaria as the % positive behaviors increased from 27 to 63 %. The results demonstrate, for the first time in planaria, scopolamine’s effects in causing learning and memory impairments and galantamine’s ability in reversing scopolamine-induced memory impairments.     

Effects of Treadmill Exercise on Neural Stem Cells, Cell Proliferation, and Neuroblast Differentiation in the Subgranular Zone of the Dentate Gyrus in Cyclooxygenase-2 Knockout Mice

Cyclooxygenase-2 (COX-2) function has been implicated in a number of physiological processes, including inflammatory responses, synaptic transmission, and synaptic plasticity in the brain. However, the specific role of COX-2 in exercise-induced neurogenesis is still debatable. Here, we assessed the role of COX-2 in exercise-induced plasticity by comparing COX-2 knockout mice to wild-type control littermates. We investigated the number of neural stem cells, and the degree of cell proliferation and neuronal differentiation in COX-2 knockout and its wild-type mice that either exercised or remained inactive. Wild-type and COX-2 knockout mice were put on a treadmill and were either sedentary or were forced to run 1 h/day for five consecutive days at a pace of 10–12 m/min for 5 weeks. Loss of COX-2 expression in the knockout mice was confirmed with two measures: (1) COX immunolabeling in the hippocampus, and (2) the identification of abnormal kidney development using hematoxylin and eosin staining, including subcapsular glomerular hypoplasia and hypertrophy of the deeper cortical glomeruli. Compared to wild-type mice, COX-2 knockout mice exhibited a significant reduction in the neural stem cells (nestin-positive cells), cell proliferation (Ki67-positive cells), and neuroblast differentiation (doublecortin-positive cells). In contrast, exercise significantly increased the neural stem cells, cell proliferation, and neuroblast differentiation in both the wild-type and COX-2 knockout mice although the NeuN-immunoreactive neurons were similar in all groups. Expression of phosphorylated cAMP-response element binding protein was decreased in knockout mice. Exercise increased its expression in the subgranular zone of the dentate gyrus in both wild-type and knockout mice. These results suggest that the COX-2 pathway is one of important factors on neural stem cells, cell proliferation and neuroblast differentiation in sedentary mice. The ability of exercise to increase these types of neural plasticity, regardless of COX-2 signaling, suggests that the effects of exercise on neural stem cells, cell proliferation, and neuroblast differentiation are induced via a pathway that is independent of COX-2.     

Lack of evidence in neurite growth in the gerbil hippocampal CA1 region 15 days after transient forebrain ischemia

Transient forebrain ischemia promotes a robust increase in neuroblast differentiation in the hippocampal dentate gyrus that peaks 7–15 days after the surgery. In this study, we compared the glucose transporter 3 (GLUT3)-dependent glucose utilization and the dynamin-1 (DNM1)-dependent neurite growth in the hippocampus of Mongolian gerbils 15 days after the induction of transient forebrain ischemia. The animals were subjected to a 5 min transient ischemia protocol and sacrificed 15 days after the surgery. Both doublecortin (DCX) immunoreactive neuroblasts and DCX total protein levels were abundantly increased in the ischemic group compared to the levels observed in the control group. In addition, animals in the ischemic group showed elevated GLUT3 immunoreactivity in the subgranular zone of the dentate gyrus compared to animals in the control group. Based on the double immunofluorescent study, increased DCX-immunoreactive neuroblasts were co-localized with GLUT3-immunoreactive components in the dentate gyrus. However, both the immunoreactivity and the total protein levels of DNM1 were significantly decreased in the dentate gyrus and hippocampal CA1 regions of the ischemic group. These results suggest that the regeneration process such as neurite growth is lacking in the hippocampus 15 days after ischemia/reperfusion although neuroblasts production and glucose utilization increased in the hippocampus.     

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

BackgroundIn the present study, we investigated the effects of pyridoxine on hippocampal functions and changes in protein profiles based on the proteomic approach.MethodsEight-week-old mice received intraperitoneal injections of physiological saline (vehicle) or 350 mg/kg pyridoxine twice a day for 21 days.ResultsPhosphoglycerate 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 3 weeks 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.ConclusionThese 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 significanceVitamin-B₆ enhances hippocampal functions and this is closely associated with CRIP1a and CB1 cannabinoid receptors.     

Chronic Effects of Pyridoxine in the Gerbil Hippocampal CA1 Region after Transient Forebrain Ischemia

In a previous study, we reported that the administration of pyridoxine (vitamin B₆) to mice for 3 weeks significantly increased cell proliferation and neuroblast differentiation in the dentate gyrus without any neuronal damage. In the present study, we investigated the restorative potentials of pyridoxine on ischemic damage in the hippocampal CA1 region of Mongolian gerbils. Gerbils were subjected to 5 min of transient ischemia, and surgical operation success was assessed by ophthalmoscope during occlusion of common carotid arteries and spontaneous motor activity at 1 day after ischemia/reperfusion. Pyridoxine (350 mg/kg) or its vehicle (physiological saline) was intraperineally administered to ischemic gerbils twice a day starting 4 days after ischemia/reperfusion for 30 or 60 days. The repeated administration of pyridoxine for 30 and 60 days significantly increased doublecortin-immunoreactive neuroblasts in the dentate gyrus and increased NeuN-immunoreactive mature neurons and βIII-tubulin-immunoreactive dendrites in the hippocampal CA1 region. Furthermore, brain-derived neurotrophic factor (BDNF) protein levels were significantly increased in pyridoxine-treated groups compared to those in the vehicle-treated groups. These results suggest that chronic administration of pyridoxine enhances neuroblast differentiation in the dentate gyrus and induces new mature neurons in the hippocampal CA1 region by up-regulating BDNF expression in hippocampal homogenates.