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.     

Testosterone and dihydrotestosterone, but not estradiol, enhance survival of new hippocampal neurons in adult male rats

Past research suggested that androgens may play a role in the regulation of adult neurogenesis within the dentate gyrus. We tested this hypothesis by manipulating androgen levels in male rats. Castrated or sham castrated male rats were injected with 5-Bromo-2'deoxyuridine (BrdU). BrdU-labeled cells in the dentate gryus were visualized and phenotyped (neural or glial) using immunohistochemistry. Castrated males showed a significant decrease in 30-day cell survival within the dentate gyrus but there was no significant change in cell proliferation relative to control males, indicating that androgens positively affect cell survival, but not cell proliferation. To examine the role of testosterone on hippocampal cell survival, males were injected with testosterone s.c. for 30 days starting the day after BrdU injection. Higher doses (0.5 and 1.0 mg/kg) but not a lower dose (0.25 mg/kg) of testosterone resulted in a significant increase in neurogenesis relative to controls. We next tested the role of testosterone's two major metabolites, dihydrotestosterone (DHT), and estradiol, upon neurogenesis. Thirty days of injections of DHT (0.25 and 0.50 mg/kg) but not estradiol (0.010 and 0.020 mg/kg) resulted in a significant increase in hippocampal neurogenesis. These results suggest that testosterone enhances hippocampal neurogenesis via increased cell survival in the dentate gyrus through an androgen-dependent mechanism.     

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.     

Estradiol initially enhances but subsequently suppresses (via adrenal steroids) granule cell proliferation in the dentate gyrus of adult female rats

In the dentate gyrus of adult female meadow voles, a high dose of estradiol benzoate (EB) increases (within 4 h) then decreases (within 48) the number of dividing progenitor cells (Ormerod BK, Galea LAM. 2001. Reproductive status regulates cell proliferation within the dentate gyrus of the adult female meadow vole: A possible regulatory role for estradiol. Neurosci 2:169-179). We investigated whether time-dependent EB exposure differentially influences the number of new granule cells produced in the adult female rat dentate gyrus and whether EB-stimulated adrenal activity mediates the decrease in cell proliferation. Ovariectomized rats received either an EB (10 microg in 0.1 mL) or vehicle (0.1 mL) injection either 4 or 48 h (Experiment 1) before a BrdU injection (200 mg/kg) and were perfused 24 h later to assess the number of new cells. Relative to vehicle, the number of new cells increased following a 4 h exposure (p < or = 0.04) but decreased following a 48 h exposure (p < or = 0.006) to EB. In Experiment 2, the number of new cells within the dentate gyrus of ovariectomized and adrenalectomized females did not significantly differ between groups exposed to EB versus vehicle for 48 h prior to BrdU administration, suggesting the decreased number of new cells observed within the dentate gyrus of adrenal-intact adult female rats is mediated by EB-stimulated adrenal activity. We conclude that estradiol dynamically regulates cell proliferation within the dentate gyrus of adult female rats in the time-dependent manner observed previously in voles and suppresses cell proliferation by influencing adrenal steroids. Investigating how estradiol dynamically regulates neurogenesis could provide insight into the mechanisms by which the proliferation of progenitor cells is controlled within the adult rodent hippocampus.     

Neuropeptide Y stimulates neuronal precursor proliferation in the post-natal and adult dentate gyrus

Adult dentate neurogenesis is important for certain types of hippocampal-dependent learning and also appears to be important for the maintenance of normal mood and the behavioural effects of antidepressants. Neuropeptide Y (NPY), a peptide neurotransmitter released by interneurons in the dentate gyrus, has important effects on mood, anxiety-related behaviour and learning and memory. We report that adult NPY receptor knock-out mice have significantly reduced cell proliferation and significantly fewer immature doublecortin-positive neurons in the dentate gyrus. We also show that the neuroproliferative effect of NPY is dentate specific, is Y1-receptor mediated and involves extracellular signal-regulated kinase (ERK)1/2 activation. NPY did not exhibit any effect on cell survival in vitro but constitutive loss of the Y1 receptor in vivo resulted in greater survival of newly generated neurons and an unchanged total number of dentate granule cells. These results show that NPY stimulates neuronal precursor proliferation in the dentate gyrus and suggest that NPY-releasing interneurons may modulate dentate neurogenesis.     

Repeated Administration of PEP-1-Cu,Zn-Superoxide Dismutase and PEP-1-Peroxiredoxin-2 to Senescent Mice Induced by d-galactose Improves the Hippocampal Functions

Oxidative stress initiates age-related reduction in hippocampal neurogenesis and the use of antioxidants has been proposed as an effective strategy to prevent or attenuate the reduction of neurogenesis in the hippocampus. In the present study, we investigated the effects of Cu,Zn-superoxide dismutase (SOD1) and/or peroxiredoxin-2 (PRX2) on cell proliferation and neuroblast differentiation in the dentate gyrus in a model of d-galactose-induced aging model. For this study, we constructed an expression vector, PEP-1, fused PEP-1 with SOD1 or PRX2, and generated PEP-1-SOD1 and PEP-1-PRX2 fusion protein. The aging model was induced by subcutaneous injection of d-galactose (100 mg/kg) to 6-week-old male mice for 10 weeks. PEP-1, PEP-1-SOD1 and/or PEP-1-PRX2 fusion protein was intraperitoneally administered to these mice at 13-week-old once a day for 3 weeks and sacrificed at 30 min after the last administrations. The administration of PEP-1-SOD1 and/or PEP-1-PRX2 significantly improved d-galactose-induced deficits on the escape latency, swimming speeds, platform crossings, spatial preference for the target quadrant in Morris water maze test. In addition, the administration of PEP-1-SOD1 and/or PEP-1-PRX2 ameliorated d-galactose-induced reductions of cell proliferation and neuroblast differentiation in the dentate gyrus and significantly reduced d-galactose-induced lipid peroxidation in the hippocampus. These effects were more prominent in the PEP-1-SOD1-treated group with PEP-1-PRX2. These results suggest that a SOD1 and/or PRX2 supplement to aged mice could improve the memory deficits, cell proliferation and neuroblast differentiation in the dentate gyrus of d-galactose induced aged mice by reducing lipid peroxidation.     

Age-dependent acute interference with stem and progenitor cell proliferation in the hippocampus after exposure to 1800 MHz electromagnetic radiation

To investigate the effects of exposure to an 1800 MHz electromagnetic field on cell death and cell proliferation in the developing brain, postnatal day 7 (P7) and P21 healthy Kunming mice were randomly assigned into the experimental and control groups. The experimental groups were exposed to an 1800 MHz electromagnetic field for 8 h daily for three consecutive days. The thymidine analog 5-bromo-2-deoxyuridine (BrdU) was injected intraperitoneally 1 h before each exposure session, and all animals were sacrificed 24 h after the last exposure. Cell death and proliferation markers were detected by immunohistochemistry in the dentate gyrus of the hippocampus. Electromagnetic exposure has no influence on cell death in the dentate gyrus of the hippocampus in P7 and P21 mice as indicated by active caspase-3 immunostaining and Fluoro-Jade labeling. The basal cell proliferation in the hippocampus was higher in P7 than in P21 mice as indicated by the number of cells labeled with BrdU and by immunohistochemical staining for phosphor-histone H3 (PHH3) and brain lipid-binding protein (BLBP). Electromagnetic exposure stimulated DNA synthesis in P7 neural stem and progenitor cells, but reduced cell division and the total number of stem cells in the hippocampus as indicated by increased BrdU labeling and reduced PHH3 and BLBP labeling compared to P7 control mice. There were no significant changes in cell proliferation in P21 mice after exposure to the electromagnetic field. These results indicate that interference with stem cell proliferation upon short-term exposure to an 1800 MHz electromagnetic field depends on the developmental stage of the brain.     

Neuronal Ras activation inhibits adult hippocampal progenitor cell division and impairs spatial short‐term memory

A large number of endogenous and exogenous factors have been identified to upregulate and downregulate proliferation, differentiation and/or survival of newborn cells in the adult hippocampus. For studying neuronal mechanisms mediating the impact of those factors, we used a transgenic synRas mouse model expressing constitutively activated Valin12‐Harvey Ras selectively in differentiated neurons. BrdU injections showed significantly reduced proliferation of new cells within the adult hippocampus of transgenic animals compared with their wild‐type siblings. In contrast, the relative survival of newborn cells was increased in synRas mice, although this effect did not fully compensate for diminished proliferation. Inhibition of progenitor cell proliferation and enhancement of cellular survival were more pronounced in males compared with females. Double labelling and doublecortin immunostaining verified that specifically newborn neurons were decreased in synRas mice. Reduced cell generation was observed already 2 h after BrdU pulse injections, identifying an early precursor cell population as target of the inhibitory transgene effect. Differences in proliferation remained stable after 24 h and were specific for the subgranular zone of the dentate gyrus, as subventricular cell generation was not affected supporting a non‐cell autonomous effect on neural hippocampal progenitors. Transgene expression only starts with synaptic differentiation and therefore reduced proliferation must represent an indirect secondary consequence of synRas activity in differentiated neurons. This was associated with impaired spatial short‐term memory capacities as observed in a radial maze paradigm. Our data suggest that constantly high Ras activity in differentiated neurons downregulates hippocampal precursor cell generation in the neuronal lineage, but is modulated by sex‐dependent factors.     

Evidence that brain-derived neurotrophic factor is required for basal neurogenesis and mediates,in part,the enhancement of neurogenesis by dietary restriction in the hippocampus of adult mice

To determine the role of brain-derived neurotrophic factor (BDNF) in the enhancement of hippocampal neurogenesis resulting from dietary restriction (DR), heterozygous BDNF knockout (BDNF +/-) mice and wild-type mice were maintained for 3 months on DR or ad libitum (AL) diets. Mice were then injected with bromodeoxyuridine (BrdU) and killed either 1 day or 4 weeks later. Levels of BDNF protein in neurons throughout the hippocampus were decreased in BDNF +/- mice, but were increased by DR in wild-type mice and to a lesser amount in BDNF +/- mice. One day after BrdU injection the number of BrdU-labeled cells in the dentate gyrus of the hippocampus was significantly decreased in BDNF +/- mice maintained on the AL diet, suggesting that BDNF signaling is important for proliferation of neural stem cells. DR had no effect on the proliferation of neural stem cells in wild-type or BDNF +/- mice. Four weeks after BrdU injection, numbers of surviving labeled cells were decreased in BDNF +/- mice maintained on either AL or DR diets. DR significantly improved survival of newly generated cells in wild-type mice, and also improved their survival in BDNF +/- mice, albeit to a lesser extent. The majority of BrdU-labeled cells in the dentate gyrus exhibited a neuronal phenotype at the 4-week time point. The reduced neurogenesis in BDNF +/- mice was associated with a significant reduction in the volume of the dentate gyrus. These findings suggest that BDNF plays an important role in the regulation of the basal level of neurogenesis in dentate gyrus of adult mice, and that by promoting the survival of newly generated neurons BDNF contributes to the enhancement of neurogenesis induced by DR.     

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.     

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.     

Enriched environment increases neurogenesis in the adult rat dentate gyrus and improves spatial memory.

The fetal and even the young brain possesses a considerable degree of plasticity. The plasticity and rate of neurogenesis in the adult brain is much less pronounced. The present study was conducted to investigate whether housing conditions affect neurogenesis, learning, and memory in adult rats. Three-month-old rats housed either in isolation or in an enriched environment were injected intraperitoneally with bromodeoxyuridine (BrdU) to detect proliferation among progenitor cells and to follow their fate in the dentate gyrus. The rats were sacrificed either 1 day or 4 weeks after BrdU injections. This experimental paradigm allows for discrimination between proliferative effects and survival effects on the newborn progenitors elicited by different housing conditions. The number of newborn cells in the dentate gyrus was not altered 1 day after BrdU injections. In contrast, the number of surviving progenitors 1 month after BrdU injections was markedly increased in animals housed in an enriched environment. The relative ratio of neurogenesis and gliogenesis was not affected by environmental conditions, as estimated by double-labeling immunofluorescence staining with antibodies against BrdU and either the neuronal marker calbindin D28k or the glial marker GFAp, resulting in a net increase in neurogenesis in animals housed in an enriched environment. Furthermore, we show that adult rats housed in an enriched environment show improved performance in a spatial learning test. The results suggest that environmental cues can enhance neurogenesis in the adult hippocampal region, which is associated with improved spatial memory.     

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.     

Increased cell proliferation in the adult mouse hippocampus following chronic administration of group II metabotropic glutamate receptor antagonist, MGS0039

We have previously reported that MGS0039, a novel antagonist of group II metabotropic glutamate receptors (mGluRs), exerts antidepressant-like effects in experimental animal models. Recent studies suggest that the behavioral effects of chronic antidepressant treatment are mediated by the stimulation of neurogenesis in the hippocampus. In the present study, we examined the effects of MGS0039 on cell proliferation in the adult mouse hippocampus. MGS0039 (5 or 10mg/kg) or fluvoxamine was administered chronically to male ICR mice over a period of 14 days. Multiple bromodeoxyuridine (BrdU) administrations were performed after the last drug injection to label dividing cells. Immunohistochemical analyses after BrdU injections revealed that chronic MGS0039 treatment enhanced BrdU-positive cells in the dentate gyrus ( approximately 62% increase) in the same manner as chronic fluvoxamine treatment. This is the first in vivo study to demonstrate an increase in cell proliferation following a blockade of group II mGluRs. These findings raise the possibility that MGS0039 may exert antidepressant-like effects by modulating cell proliferation in the hippocampus.     

Enriched environment increases neurogenesis in the adult rat dentate gyrus and improves spatial memory

The fetal and even the young brain possesses a considerable degree of plasticity. The plasticity and rate of neurogenesis in the adult brain is much less pronounced. The present study was conducted to investigate whether housing conditions affect neurogenesis, learning, and memory in adult rats. Three‐month‐old rats housed either in isolation or in an enriched environment were injected intraperitoneally with bromodeoxyuridine (BrdU) to detect proliferation among progenitor cells and to follow their fate in the dentate gyrus. The rats were sacrificed either 1 day or 4 weeks after BrdU injections. This experimental paradigm allows for discrimination between proliferative effects and survival effects on the newborn progenitors elicited by different housing conditions. The number of newborn cells in the dentate gyrus was not altered 1 day after BrdU injections. In contrast, the number of surviving progenitors 1 month after BrdU injections was markedly increased in animals housed in an enriched environment. The relative ratio of neurogenesis and gliogenesis was not affected by environmental conditions, as estimated by double‐labeling immunofluorescence staining with antibodies against BrdU and either the neuronal marker calbindin D28k or the glial marker GFAp, resulting in a net increase in neurogenesis in animals housed in an enriched environment. Furthermore, we show that adult rats housed in an enriched environment show improved performance in a spatial learning test. The results suggest that environmental cues can enhance neurogenesis in the adult hippocampal region, which is associated with improved spatial memory. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 569–578, 1999     

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.     

Systemic Inflammation Impairs Proliferation of Hippocampal Type 2 Intermediate Precursor Cells

Neurogenesis is a plastic event modulated by external cues. Systemic inflammation decreases neurogenesis in the dentate gyrus (DG) in part through the proliferative restrain of neural precursor cells (NPCs). To evaluate if inflammation affects the cell cycle progression of particular populations of NPCs, we treated young-adult mice with a single i.p. injection of saline or 1 mg/kg LPS. After 7 days, we analysed proliferation of new BrdU+/DCX+ cells through immunohistochemistry. We extracted the hippocampus and performed a neurosphere assay and a flow cytometric analysis to evaluate proliferation and to identify the phase of the cell cycle in specific populations of DG-derived NPCs. We show that the number of BrdU+/DCX+ cells diminishes in the LPS-treated group and that the number of primary neurospheres derived from LPS-injected animals is significantly reduced compared to the saline-injected group. Flow cytometry revealed that inflammation does not affect the total number of Type 1 BLBP+/TBR2? cells, while the total number of Type 2 intermediate precursor cells (IPCs) (TBR2+) from the LPS-treated group was increased. Cell cycle analysis shows a decrease in the total rate of NPCs in phases S, G2 and M in the LPS-treated group. The percentage of Type 1 BLBP+/TBR2? cells in each cell cycle phase was not different between groups, while there was a fewer number of Type 2 TBR2+ cells in S/G2/M phase. These results show that inflammation alters the appropriate cell cycle progression of Type 2 IPCs, which may contribute to the decrease in the birth rate of DG neurons.     

Neuroprotective effects of quercetin, rutin and okra (Abelmoschus esculentus Linn.) in dexamethasone-treated mice

The administration of dexamethasone, a synthetic glucocorticoid receptor agonist, causes neuronal death in the CA3 layer of the hippocampus, which has been associated with learning and memory impairments. This study aimed to examine the ability of okra (Abelmoschus esculentus Linn.) extract and its derivatives (quercetin and rutin) to protect neuronal function and improve learning and memory deficits in mice subjected to dexamethasone treatment. Learning and memory functions in mice were examined using the Morris water maze test. The results showed that the mice treated with dexamethasone had prolonged water maze performance latencies and shorter time spent in the target quadrant while mice pretreated with quercetin, rutin or okra extract prior to dexamethasone treatment showed shorter latencies and longer time spent in target quadrant. Morphological changes in pyramidal neurons were observed in the dexamethasone treated group. The number of CA3 hippocampal neurons was significantly lower while pretreated with quercetin, rutin or okra attenuated this change. Prolonged treatment with dexamethasone altered NMDA receptor expression in the hippocampus. Pretreatment with quercetin, rutin or okra extract prevented the reduction in NMDA receptor expression. Dentate gyrus (DG) cell proliferation was examined using the 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry technique. The number of BrdU-immunopositive cells was significantly reduced in dexamethasone-treated mice compared to control mice. Pretreatment with okra extract, either quercetin or rutin was found to restore BrdU-immunoreactivity in the dentate gyrus. These findings suggest that quercetin, rutin and okra extract treatments reversed cognitive deficits, including impaired dentate gyrus (DG) cell proliferation, and protected against morphological changes in the CA3 region in dexamethasone-treated mice. The precise mechanism of the neuroprotective effect of these plant extracts should be further investigated.