Natural antioxidant formula ameliorates lipopolysaccharide-induced impairment of hippocampal neurogenesis and contextual fear memory through suppression of neuroinflammation in rats.

This study investigated the ameliorating effects of a natural antioxidant formula (NAF) consisting of Ginkgo biloba leaf extract, docosahexaenoic acid/eicosapentaenoic acid, ferulic acid, flaxseed oil, vitamin E, and vitamin B12 on a lipopolysaccharide (LPS)-induced cognitive dysfunction model in rats. Six-week-old rats received a diet containing 0.5% (w/w) NAF for 38 days from Day 1, and LPS (1 mg/kg body weight) was administered intraperitoneally once daily on Days 8 and 10. On Day 11, LPS alone increased interleukin-1ß and tumor necrosis factor-α in the hippocampus and cerebral cortex and the numbers of M1-type microglia/macrophages and GFAP+ reactive astrocytes in the hilus of the hippocampal dentate gyrus. NAF treatment decreased brain proinflammatory cytokine levels and increased the number of M2-type microglia/macrophages. During Days 34-38, LPS alone impaired fear memory acquisition and the extinction learning process, and NAF facilitated fear extinction learning. On Day 38, LPS alone decreased the number of type-3 neural progenitor cells in the hippocampal neurogenic niche, and NAF restored the number of type-3 neural progenitor cells and increased the numbers of both immature granule cells in the neurogenic niche and reelin+ hilar interneurons. Thus, NAF exhibited anti-inflammatory effects and ameliorated LPS-induced adverse effects on hippocampal neurogenesis and fear memory learning, possibly through amplification of reelin signaling by hilar interneurons. These results suggest that neuroinflammation is a key factor in the development of LPS-induced impairment of fear memory learning, and supplementation with NAF in the present study helped to prevent hippocampal neurogenesis and disruptive neurobehaviors caused by neuroinflammation.

Amelioration of lipopolysaccharides-induced impairment of fear memory acquisition by alpha-glycosyl isoquercitrin through suppression of neuroinflammation in rats.

This study investigated the role of neuroinflammation in a lipopolysaccharides (LPS)-induced cognitive dysfunction model in rats using an antioxidant, α-glycosyl isoquercitrin (AGIQ). Six-week-old rats were dietary treated with 0.5% (w/w) AGIQ for 38 days, and LPS at 1 mg/kg body weight was administered intraperitoneally once daily on Days 8 and 10. On Day 11, LPS alone increased or tended to increase interleukin-1ß and tumor necrosis factor-α in the hippocampus and cerebral cortex. Immunohistochemically, LPS alone increased the number of Iba1+ and CD68+ microglia, and GFAP+ astrocytes in the hilus of the hippocampal dentate gyrus (DG). AGIQ treatment decreased or tended to decrease brain proinflammatory cytokine levels and the number of CD68+ microglia in the DG hilus. In the contextual fear conditioning test during Day 34 and Day 38, LPS alone impaired fear memory acquisition, and AGIQ tended to recover this impairment. On Day 38, LPS alone decreased the number of DCX+ cells in the neurogenic niche, and AGIQ increased the numbers of PCNA+ cells in the subgranular zone and CALB2+ hilar interneurons. Additionally, LPS alone decreased or tended to decrease the number of synaptic plasticity-related FOS+ and COX2+ granule cells and AGIQ recovered them. The results suggest that LPS administration induced acute neuroinflammation and subsequent impairment of fear memory acquisition caused by suppressed synaptic plasticity of newborn granule cells following disruptive neurogenesis. In contrast, AGIQ exhibited anti-inflammatory effects and ameliorated LPS-induced adverse effects. These results suggest that neuroinflammation is a key factor in the development of LPS-induced impairment of fear memory acquisition.

Continuous exposure to alpha-glycosyl isoquercitrin from mid-gestation ameliorates polyinosinic-polycytidylic acid-disrupted hippocampal neurogenesis in rats.

Polyinosinic-polycytidylic acid (PIC) provides a model of developmental neuropathy by inducing maternal immune activation. We investigated the effects of an antioxidant, alpha-glycosyl isoquercitrin (AGIQ), on PIC-induced developmental neuropathy in rats, focusing on postnatal hippocampal neurogenesis. On gestational day 15, PIC at 4 mg/kg body weight was administered to dams intravenously. AGIQ either at 0.25% or 0.5% was administered through the diet to dams from gestational day 10 until weaning on day 21 post-delivery and, thereafter, to offspring until postnatal day 77 (adult stage). At weaning, the numbers of TBR2+ cells and PCNA+ cells in the subgranular zone and reelin+ cells in the dentate gyrus hilus in offspring of dams treated with PIC only were decreased compared with untreated controls. In contrast, 0.5% AGIQ ameliorated these changes and increased the transcript levels of genes related to signaling of reelin (Reln and Vldlr), growth factors (Bdnf, Cntf, Igf1, and Igf1r), and Wnt/ß-catenin (Wnt5a, Lrp6, Fzd1, and Fzd3). In adults, AGIQ increased the number of FOS+ granule cells at 0.25% and the transcript levels of NMDA-type glutamate receptor genes, Grin2a and Grin2b, at 0.25% and 0.5%, respectively. These results suggest that mid-gestation PIC treatment decreased the abundance of type-2b neural progenitor cells (NPCs) by reducing NPC proliferation in relation with suppression of reelin signaling at weaning. We suggest that AGIQ ameliorated the PIC-induced suppressed neurogenesis by enhancing reelin, growth factor, and Wnt/ß-catenin signaling at weaning to rescue NPC proliferation and increased synaptic plasticity by enhancing glutamatergic signaling via NMDA-type receptors after maturation.

Comparison of the effect of glyphosate and glyphosate-based herbicide on hippocampal neurogenesis after developmental exposure in rats.

Increasing evidence indicates that glyphosate (GlyP)-based herbicides (GBHs) induce developmental neurotoxicity. The present study investigated the developmental exposure effect of GlyP and GBH on hippocampal neurogenesis in rats. Dams were treated from gestational day 6 to day 21 post-delivery on weaning with a diet containing 1.5% or 3.0% GlyP or drinking water with 1.0% GBH (containing 0.36% GlyP). Dams in the 1.5%-GlyP, 3.0%-GlyP, and GBH groups received 1.04, 2.16, and 0.25 g GlyP/kg body weight (BW)/day during gestation, and 2.27, 4.65, and 0.58 g GlyP/kg BW/day during lactation, respectively. On weaning, 3.0% GlyP- and GBH-exposed offspring decreased the BW, and the latter also decreased the brain weight. Both compounds suppressed neural progenitor cell proliferation in the neurogenic niche, and GlyP-exposed offspring showed a decreased number of TUBB3+ immature granule cells. In contrast, both compounds increased the number of ARC+ granule cells, suggesting increased synaptic plasticity. Both compounds downregulated antioxidant genes (Cat and Sod2) in the dentate gyrus, suggestive of increased sensitivity to oxidative stress, which might be related to the suppression of neurogenesis. At the adult age, GBH alone sustained decreases in body and brain weights. Both compounds increased hippocampal malondialdehyde levels and upregulated Cat in the dentate gyrus, suggesting induction of oxidative stress. Both compounds upregulated Casp9, and GBH increased neural progenitor cell apoptosis, suggesting disruption of neurogenesis related to oxidative stress. GBH increased the number of COX2+ granule cells, and both compounds upregulated Arc, suggesting increased synaptic plasticity. These results suggest that GlyP and GBH might cause similar effects on disruption of neurogenesis accompanying compensatory responses and induction of oxidative stress responses through the adult age in the hippocampus. However, effects on adult age were more evident with GBH, suggesting that the surfactants contained in GBH might have contributed to the enhanced neurotoxicity of GlyP, similar to the enhanced general toxicity.

Progressive disruption of neurodevelopment by mid-gestation exposure to lipopolysaccharides and the ameliorating effect of continuous alpha-glycosyl isoquercitrin treatment.

We investigated the effect of lipopolysaccharide (LPS)-induced maternal immune activation used as a model for producing neurodevelopmental disorders on hippocampal neurogenesis and behaviors in rat offspring by exploring the antioxidant effects of alpha-glycosyl isoquercitrin (AGIQ). Pregnant Sprague-Dawley rats were intraperitoneally injected with LPS (50 µg/kg body weight) at gestational days 15 and 16. AGIQ was administered in the diet to dams at 0.5% (w/w) from gestational day 10 until weaning at postnatal day 21 and then to offspring until adulthood at postnatal day 77. During postnatal life, offspring of LPS-injected animals did not show neuroinflammation or oxidative stress in the brain. At weaning, LPS decreased the numbers of type-2b neural progenitor cells (NPCs) and PCNA+ proliferating cells in the subgranular zone, FOS-expressing granule cells, and GAD67+ hilar interneurons in the dentate gyrus. In adulthood, LPS decreased type-1 neural stem cells, type-2a NPCs, and GAD67+ hilar interneurons, and downregulated Dpysl3, Sst, Fos, Mapk1, Mapk3, Grin2a, Grin2b, Bdnf, and Ntrk2. In adults, LPS suppressed locomotor activity in the open field test and suppressed fear memory acquisition and fear extinction learning in the contextual fear conditioning test. These results indicate that mid-gestation LPS injections disrupt programming of normal neurodevelopment resulting in progressive suppression of hippocampal neurogenesis and synaptic plasticity of newborn granule cells by suppressing GABAergic and glutamatergic neurotransmitter signals and BDNF/TrkB signaling to result in adult-stage behavioral deficits. AGIQ ameliorated most aberrations in hippocampal neurogenesis and synaptic plasticity, as well as behavioral deficits. Effective amelioration by continuous AGIQ treatment starting before LPS injections may reflect both anti-inflammatory and anti-oxidative stress effects during gestation and neuroprotective effects of continuous exposure through adulthood.

Oral Exposure to Lead Acetate for 28 Days Reduces the Number of Neural Progenitor Cells but Increases the Number and Synaptic Plasticity of Newborn Granule Cells in Adult Hippocampal Neurogenesis of Young-Adult Rats.

Lead (Pb) causes developmental neurotoxicity. Developmental exposure to Pb acetate (PbAc) induces aberrant hippocampal neurogenesis by increasing or decreasing neural progenitor cell (NPC) subpopulations in the dentate gyrus (DG) of rats. To investigate whether hippocampal neurogenesis is similarly affected by PbAc exposure in a general toxicity study, 5-week-old Sprague-Dawley rats were orally administered PbAc at 0, 4000, and 8000 ppm (w/v) in drinking water for 28 days. After exposure to 4000 or 8000 ppm PbAc, Pb had accumulated in the brains. Neurogenesis was suppressed by 8000 ppm PbAc, which was related to decreased number of type-2b NPCs, although number of mature granule cells were increased by both PbAc doses. Gene expression in the 8000 ppm PbAc group suggested suppressed NPC proliferation and increased apoptosis resulting in suppressed neurogenesis. PbAc exposure increased numbers of metallothionein-I/II+ cells and GFAP+ astrocytes in the DG hilus, and upregulated Mt1, antioxidant genes (Hmox1 and Gsta5), and Il6 in the DG, suggesting the induction of oxidative stress and neuroinflammation related to Pb accumulation resulting in suppressed neurogenesis. PbAc at 8000 ppm also upregulated Ntrk2 and increased the number of CALB2+ interneurons, suggesting the activation of BDNF-TrkB signaling and CALB2+ interneuron-mediated signals to ameliorate suppressed neurogenesis resulting in increased number of newborn granule cells. PbAc at both doses increased the number of ARC+ granule cells, suggesting the facilitation of synaptic plasticity of newborn granule cells through the activation of BDNF-TrkB signaling. These results suggest that PbAc exposure during the young-adult stage disrupted hippocampal neurogenesis, which had a different pattern from developmental exposure to PbAc. However, the induction of oxidative stress/neuroinflammation and activation of identical cellular signals occurred irrespective of the life stage at PbAc exposure.

Continuous Exposure to Alpha-Glycosyl Isoquercitrin from Gestation Ameliorates Disrupted Hippocampal Neurogenesis in Rats Induced by Gestational Injection of Valproic Acid.

This study examined the ameliorating effect of alpha-glycosyl isoquercitrin (AGIQ), an antioxidant, on disrupted hippocampal neurogenesis in the dentate gyrus (DG) in a rat model of autism spectrum disorder induced by prenatal valproic acid (VPA) exposure. Dams were intraperitoneally injected with 500 mg/kg VPA on gestational day 12. AGIQ was administered in the diet at 0.25 or 0.5% to dams from gestational day 13 until weaning at postnatal day (PND) 21 and then to pups until PND 63. At PND 21, VPA-exposed offspring showed decreased numbers of type-2a and type-3 neural progenitor cells (NPCs) among granule cell lineage subpopulations. AGIQ treatment at both doses rescued the reduction in type-3 NPCs. AGIQ upregulated Reln and Vldlr transcript levels in the DG at 0.5% and ≥ 0.25%, respectively, and increased the number of reelin+ interneurons in the DG hilus at 0.5%. AGIQ at 0.25% and/or 0.5% also upregulated Ntrk2, Cntf, Igf1, and Chrnb2. At PND 63, there were no changes in the granule cell lineage subpopulations in response to VPA or AGIQ. AGIQ at 0.25% increased the number of FOS+ granule cells, accompanied by Gria2 and Gria3 upregulation and increasing trend in the number of FOS+ granule cells at 0.5%. There was no definitive evidence of VPA-induced oxidative stress in the hippocampus throughout postnatal life. These results indicate that AGIQ ameliorates the VPA-induced disruption of hippocampal neurogenesis at weaning involving reelin, BDNF-TrkB, CNTF, and IGF1 signaling, and enhances FOS-mediated synaptic plasticity in adulthood, potentially through AMPA-receptor upregulation. The ameliorating effects of AGIQ may involve direct interactions with neural signaling cascades rather than antioxidant capacity.

Oral exposure to high-dose ethanol for 28 days in rats reduces neural stem cells and immediate nascent neural progenitor cells as well as FOS-expressing newborn granule cells in adult hippocampal neurogenesis.

Drinking alcohol during pregnancy may cause fetal alcohol spectrum disorder. In rats, developmental exposure to ethanol (EtOH) at high doses has shown to induce aberrant neurogenesis in neural progenitor cells (NPCs) during weaning and suppress synaptic plasticity of newborn granule cells after maturation; neuroinflammation was even sustained until the adult stage in the hippocampal dentate gyrus (DG). To investigate whether hippocampal neurogenesis is affected by EtOH exposure in a general toxicity study, EtOH was administered orally to 5-week-old Sprague-Dawley rats at 0%, 10%, and 16% (w/v) in drinking water for 28 days. Exposure to 16% EtOH decreased type-1 neural stem cells (NSCs) and type-2a NPCs in the DG subgranular zone. A reduction in reelin-positive (reelin+) interneurons and an increased number of parvalbumin+ interneurons in the DG hilus, as well as downregulation of Mcm6 and Calb2 in the DG, suggested that self-renewal and proliferation of type-1 NSCs were suppressed. Exposure to 16% EtOH also induced M1-type microglia/peripheral macrophages, and upregulated Il1a and Tnf, suggesting that neuroinflammation might be responsible for the suppressed neurogenesis. In contrast, Drd2 and Tgfb3 upregulation might be ameliorating responses against suppressed neurogenesis. EtOH exposure (16%) also decreased the number of FOS+ granule cells, suggesting that synaptic plasticity was suppressed; concurrent upregulation of glutamate receptor/transporter genes may have occurred as a compensatory response against suppressed synaptic plasticity. Thus, high-dose EtOH exposure in young adult rats disrupted hippocampal neurogenesis differently to exposure during development. However, induction of neuroinflammation and suppressed synaptic plasticity occurred at both EtOH exposure stages.

Oral exposure to aluminum chloride for 28 days suppresses neural stem cell proliferation and increases mature granule cells in adult hippocampal neurogenesis of young-adult rats.

Aluminum (Al), a common light metal, affects the developing nervous system. Developmental exposure to Al chloride (AlCl3 ) induces aberrant neurogenesis by targeting neural stem cells (NSCs) and/or neural progenitor cells (NPCs) in the dentate gyrus (DG) of rats and mice. To investigate whether hippocampal neurogenesis is similarly affected by AlCl3 exposure in a general toxicity study, AlCl3 was orally administered to 5-week-old Sprague Dawley rats at dosages of 0, 4000, or 8000 ppm in drinking water for 28 days. AlCl3 downregulated Sox2 transcript level in the DG at the highest dosage and produced a dose-dependent decrease of SOX2+ cells without altering numbers of GFAP+ or TBR2+ cells in the subgranular zone, suggesting that AlCl3 decreases Type 2a NPCs. High-dose exposure downregulated Pcna, upregulated Pvalb, and altered expression of genes suggestive of oxidative stress induction (upregulation of Nos2 and downregulation of antioxidant enzyme genes), indicating suppressed proliferation and differentiation of Type 1 NSCs. AlCl3 doses also increased mature granule cells in the DG. Upregulation of Reln may have contributed to an increase of granule cells to compensate for the decrease of Type 2a NPCs. Moreover, upregulation of Calb2, Gria2, Mapk3, and Tgfb3, as well as increased numbers of activated astrocytes in the DG hilus, may represent ameliorating responses against suppressed neurogenesis. These results suggest that 28-day exposure of young-adult rats to AlCl3 differentially targeted NPCs and mature granule cells in hippocampal neurogenesis, yielding a different pattern of disrupted neurogenesis from developmental exposure.

Gene expression profiles of multiple brain regions in rats differ between developmental and postpubertal exposure to valproic acid.

We have previously reported that the valproic acid (VPA)-induced disruption pattern of hippocampal adult neurogenesis differs between developmental and 28-day postpubertal exposure. In the present study, we performed brain region-specific global gene expression profiling to compare the profiles of VPA-induced neurotoxicity between developmental and postpubertal exposure. Offspring exposed to VPA at 0, 667, and 2000 parts per million (ppm) via maternal drinking water from gestational day 6 until weaning (postnatal day 21) were examined, along with male rats orally administered VPA at 0, 200, and 900 mg/kg body weight for 28 days starting at 5 weeks old. Four brain regions-the hippocampal dentate gyrus, corpus callosum, cerebral cortex, and cerebellar vermis-were subjected to expression microarray analysis. Profiled data suggested a region-specific pattern of effects after developmental VPA exposure, and a common pattern of effects among brain regions after postpubertal VPA exposure. Developmental VPA exposure typically led to the altered expression of genes related to nervous system development (Msx1, Xcl1, Foxj1, Prdm16, C3, and Kif11) in the hippocampus, and those related to nervous system development (Neurod1) and gliogenesis (Notch1 and Sox9) in the corpus callosum. Postpubertal VPA exposure led to the altered expression of genes related to neuronal differentiation and projection (Cd47, Cyr61, Dbi, Adamts1, and Btg2) in multiple brain regions. These findings suggested that neurotoxic patterns of VPA might be different between developmental and postpubertal exposure, which was consistent with our previous study. Of note, the hippocampal dentate gyrus might be a sensitive target of developmental neurotoxicants after puberty.

Ameliorating effect of continuous alpha-glycosyl isoquercitrin treatment starting from late gestation in a rat autism model induced by postnatal injection of lipopolysaccharides.

The present study investigated the role of neuroinflammation and brain oxidative stress induced by neonatal treatment with lipopolysaccharides (LPS) on the development of autism spectrum disorder (ASD)-like behaviors and disruptive hippocampal neurogenesis in rats by exploring the chemopreventive effects of alpha-glycosyl isoquercitrin (AGIQ) as an antioxidant. AGIQ was dietary administered to dams at 0.25% or 0.5% (w/w) from gestational day 18 until postnatal day (PND) 21 on weaning and then to pups until the adult stage on PND 77. The pups were intraperitoneally injected with LPS (1 mg/kg body weight) on PND 3. At PND 6, LPS alone increased Iba1+ and CD68+ cell numbers without changing the CD163+ cell number and strongly upregulated pro-inflammatory cytokine gene expression (Il1a, Il1b, Il6, Nfkb1, and Tnf) in the hippocampus, and increased brain malondialdehyde levels. At PND 10, pups decreased ultrasonic vocalization (USV), suggesting the induction of pro-inflammatory responses and oxidative stress to trigger communicative deficits. By contrast, LPS alone upregulated Nfe2l2 expression at PND 6, increased Iba1+, CD68+, and CD163+ cell numbers, and upregulated Tgfb1 at PND 21, suggesting anti-inflammatory responses until the weaning period. However, LPS alone disrupted hippocampal neurogenesis at weaning and suppressed social interaction parameters and rate of freezing time at fear acquisition and extinction during the adolescent stage. On PND 77, neuroinflammatory responses had mostly disappeared; however, disruptive neurogenesis and fear memory deficits were sustained. AGIQ ameliorated most changes on acute pro-inflammatory responses and oxidative stress at PND 6, and the effects on USVs at PND 10 and neurogenesis and behavioral parameters throughout the adult stage. These results suggested that neonatal LPS treatment induced acute but transient neuroinflammation, triggering the progressive disruption of hippocampal neurogenesis leading to abnormal behaviors in later life. AGIQ treatment was effective for ameliorating LPS-induced progressive changes by critically suppressing initial pro-inflammatory responses and oxidative stress.

Aberrant neurogenesis and late onset suppression of synaptic plasticity as well as sustained neuroinflammation in the hippocampal dentate gyrus after developmental exposure to ethanol in rats.

Drinking alcohol during pregnancy may cause fetal alcohol spectrum disorder. The present study investigated the effects of maternal oral ethanol (EtOH) exposure (0, 10, or 12.5 % in drinking water) from gestational day 6 until day 21 post-delivery (weaning) on postnatal hippocampal neurogenesis at weaning and in adulthood on postnatal day 77 in rat offspring. At weaning, type-3 neural progenitor cells (NPCs) were decreased in the subgranular zone (SGZ), accompanied by Chrnb2 downregulation and Grin2b upregulation in the dentate gyrus (DG). These results suggested suppression of CHRNB2-mediated cholinergic signaling in γ-aminobutyric acid (GABA)ergic interneurons in the DG hilus and increased glutamatergic signaling through the NR2B subtype of N-methyl-d-aspartate (NMDA) receptors, resulting in NPC reduction. In contrast, upregulation of Chrna7 may increase CHRNA7-mediated cholinergic signaling in immature granule cells, and upregulation of Ntrk2 may cause an increase in somatostatin-immunoreactive (+) GABAergic interneurons, suggesting a compensatory response against NPC reduction. Promotion of SGZ cell proliferation increased type-2a NPCs. Moreover, an increase in calbindin-d-29 K+ interneurons and upregulation of Reln, Drd2, Tgfb2, Il18, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunit genes might participate in this compensatory response. In adulthood, reduction of FOS+ cells and downregulation of Fos and Arc suggested suppression of granule cell synaptic plasticity, reflecting upregulation of Tnf and downregulation of Cntf, Ntrk2, and AMPA-type glutamate receptor genes. In the DG hilus, gliosis and hyper-ramified microglia, accompanying upregulation of C3, appeared at weaning, suggesting contribution to suppressed synaptic plasticity in adulthood. M1 microglia increased throughout adulthood, suggesting sustained neuroinflammation. These results indicate that maternal EtOH exposure temporarily disrupts hippocampal neurogenesis and later suppresses synaptic plasticity. Induction of neuroinflammation might initially ameliorate neurogenesis (as evident by upregulation of Tgfb2 and Il18) but later suppress synaptic plasticity (as evident by upregulation of C3 at weaning and Tnf in adulthood).

Disruption of postnatal neurogenesis and adult-stage suppression of synaptic plasticity in the hippocampal dentate gyrus after developmental exposure to sterigmatocystin in rats.

Exposure to sterigmatocystin (STC) raises concerns on developmental neurological disorders. The present study investigated the effects of maternal oral STC exposure on postnatal hippocampal neurogenesis of offspring in rats. Dams were exposed to STC (1.7, 5.0, and 15.0 ppm in diet) from gestational day 6 until day 21 post-delivery (weaning), and offspring were maintained without STC exposure until adulthood on postnatal day (PND) 77, in accordance with OECD chemical testing guideline Test No. 426. On PND 21, 15.0-ppm STC decreased type-3 neural progenitor cell numbers in the subgranular zone (SGZ) due to suppressed proliferation. Increased γ-H2AX-immunoreactive (+) cell numbers in the SGZ and Ercc1 upregulation and Brip1 downregulation in the dentate gyrus suggested induction of DNA double-strand breaks in SGZ cells. Upregulation of Apex1 and Ogg1 and downregulation of antioxidant genes downstream of NRF2-Keap1 signaling suggested induction of oxidative DNA damage. Increased p21WAF1/CIP1+ SGZ cell numbers and suppressed cholinergic signaling through CHRNB2-containing receptors in GABAergic interneurons suggested potential neurogenesis suppression mechanisms. Multiple mechanisms involving N-methyl-d-aspartate (NMDA) receptor-mediated glutamatergic signaling and various GABAergic interneuron subpopulations, including CHRNA7-expressing somatostatin+ interneurons activated by BDNF-TrkB signaling, may be involved in ameliorating the neurogenesis. Upregulation of Arc, Ptgs2, and genes encoding NMDA receptors and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors suggested synaptic plasticity facilitation. On PND 77, ARC+ granule cells decreased, and Nos2 was upregulated following 15.0 ppm STC exposure, suggesting oxidative stress-mediated synaptic plasticity suppression. Inverse pattern in gene expression changes in vesicular glutamate transporter isoforms, Slc17a7 and Slc17a6, from weaning might also be responsible for the synaptic plasticity suppression. The no-observed-adverse-effect level of maternal oral STC exposure for offspring neurogenesis was determined to be 5.0 ppm, translating to 0.34-0.85 mg/kg body weight/day.

Induction of cellular senescence as a late effect and BDNF-TrkB signaling-mediated ameliorating effect on disruption of hippocampal neurogenesis after developmental exposure to lead acetate in rats.

Lead (Pb) exposure causes cognitive deficits in children. The present study investigated the effect of developmental exposure to Pb acetate (PbAc) on postnatal hippocampal neurogenesis. Pregnant rats were administered drinking water containing 0, 2000, or 4000 ppm PbAc from gestational day 6 until day 21 post-delivery (weaning), and offspring were maintained without PbAc exposure until adulthood on postnatal day (PND) 77. There was a dose-related accumulation of Pb in the offspring brain at weaning, while Pb was mainly excreted in adulthood. In the hippocampus, metallothionein I/II immunoreactive (+) glia were increased through adulthood as a neuroprotective response to accumulated Pb, accompanied by increased astrocyte and microglia numbers in adulthood, suggesting sustained neural damage. Gene expression changes suggested elevated oxidative stress at weaning and suppression of the antioxidant system in adulthood, as well as continued neuroinflammatory responses. At weaning, granule cell apoptosis was increased and numbers of type-3 neural progenitor cells (NPCs) were decreased. By contrast, type-2a and type-2b NPCs were increased, suggesting suppressed differentiation to type-3 NPCs. In adulthood, there were increased numbers of immature granule cells. In the hilus of the dentate gyrus, somatostatin+ interneurons were increased at weaning, while calbindin-D-29K+ interneurons were increased throughout adulthood, suggesting a strengthened interneuron regulatory system against the suppressed differentiation at weaning. In the dentate gyrus, Bdnf, Ntrk2, and Chrna7 gene expression were upregulated and numbers of hilar TrkB+ interneurons increased at weaning. These findings suggest activation of BDNF-TrkB signaling to increase somatostatin+ interneurons and promote cholinergic signaling, thus increasing later production of immature granule cells. In adulthood, Pcna and Apex1 gene expression were downregulated and Chek1 and cyclin-dependent kinase inhibitor expression were upregulated. Furthermore, there was an increase in γ-H2AX+ SGZ cells, suggesting induction of cellular senescence of SGZ cells due to Pb genotoxicity.

A 28-day repeated oral dose toxicity study of enniatin complex in mice.

Enniatins are so-called "emerging mycotoxins" that commonly occur in milligrams per kilogram levels in grains and their derived products, as well as in fish, dried fruits, nuts, spices, cocoa, and coffee. The present study investigated the 28-day repeated oral dose toxicity of enniatin complex in CD1(ICR) mice. Enniatin B, enniatin B1, and enniatin A1 at a ratio of 4:4:1 were administered to male and female mice at doses of 0 (vehicle controls), 0.8, 4, and 20 mg/kg body weight/day. In life parameters did not change during the study period, with the exception of slight reductions in food consumption in male mice administered 4 and 20 mg/kg and in female mice administered 20 mg/kg. Body and organ weights did not change, and no alterations in hematology, blood biochemistry, or histopathology parameters were observed at the end of the administration period. Thus, we determined that the no-observed-adverse-effect level of enniatin complex was 20 mg/kg/day for both sexes under the present experimental conditions.

Continuous exposure to amorphous formula of curcumin from the developmental stage facilitates anti-anxiety-like behavior and fear-extinction learning in rats.

An amorphous formula of curcumin (CUR) has shown to enable an improved bioavailability after ingestion. The aim of this study was to investigate the hypothesis that exogenously administered CUR has an advantage in ameliorating post-traumatic stress disorder at low doses. To this end, Long-Evans rats were dietary exposed to CUR at 0.1% or 0.5% from gestational day 6 to postnatal day (PND) 74 or 77. Offspring exposed to 0.1% CUR revealed facilitation of anti-anxiety-like behavior in the open field test and fear-extinction learning tested during PND 62 to 74, increases in hippocampal granule cells expressing immediate-early gene proteins and a decrease in prelimbic cortical neurons expressing phosphorylated extracellular signal-regulated kinase 1/2 after the last trial of the fear-extinction learning test on PND 74. The constitutive gene expression levels of Gria1, Gria2, Grin2d, Slc17a6, and Slc17a7 were altered in the hippocampal dentate gyrus and amygdala on PND 77. These results suggest alterations in synaptic plasticity to strengthen neural circuits in promoting the behavioral effects by 0.1%-CUR. In contrast, 0.5% CUR revealed a lack of any of the changes in behavioral tests that were observed at 0.1%; however, this dose upregulated oxidative stress and neuroinflammation-related genes in the hippocampal dentate gyrus, and increased neural stem cells and proliferation activity of the subgranular zone in the dentate gyrus. These results suggest a possible preventive use of CUR at low doses in mitigating some stress disorders; however, excessively absorbed doses may prevent behavioral changes by inducing neuroinflammation that affects hippocampal neurogenesis involving neural stem cells.

Identification of gene targets of developmental neurotoxicity focusing on DNA hypermethylation involved in irreversible disruption of hippocampal neurogenesis in rats.

We have previously found that maternal exposure to 6-propyl-2-thiouracil (PTU), valproic acid (VPA), or glycidol (GLY) has a sustained or late effect on hippocampal neurogenesis at the adult stage in rat offspring. Herein, we searched for genes with hypermethylated promoter region and downregulated transcript level to reveal irreversible markers of developmental neurotoxicity. The hippocampal dentate gyrus of male rat offspring exposed maternally to PTU, VPA, or GLY was subjected to Methyl-Seq and RNA-Seq analyses on postnatal day (PND) 21. Among the genes identified, 170 were selected for further validation analysis of gene expression on PND 21 and PND 77 by real-time reverse transcription-PCR. PTU and GLY downregulated many genes on PND 21, reflecting diverse effects on neurogenesis. Furthermore, genes showing sustained downregulation were found after PTU or VPA exposure, reflecting a sustained or late effect on neurogenesis by these compounds. In contrast, such genes were not observed with GLY, probably because of the reversible nature of the effects. Among the genes showing sustained downregulation, Creb, Arc, and Hes5 were concurrently downregulated by PTU, suggesting an association with neuronal mismigration, suppressed synaptic plasticity, and reduction in neural stem and progenitor cells. Epha7 and Pvalb were also concurrently downregulated by PTU, suggesting an association with the reduction in late-stage progenitor cells. VPA induced sustained downregulation of Vgf and Dpysl4, which may be related to the aberrations in synaptic plasticity. The genes showing sustained downregulation may be irreversible markers of developmental neurotoxicity.

Continuous exposure to α-glycosyl isoquercitrin from developmental stages to adulthood is necessary for facilitating fear extinction learning in rats.

We previously reported that exposure to α-glycosyl isoquercitrin (AGIQ) from the fetal stage to adulthood facilitated fear extinction learning in rats. The present study investigated the specific AGIQ exposure period sufficient for inducing this behavioral effect. Rats were dietarily exposed to 0.5% AGIQ from the postweaning stage to adulthood (PW-AGIQ), the fetal stage to postweaning stage (DEV-AGIQ), or the fetal stage to adulthood (WP-AGIQ). Fear memory, anxiety-like behavior, and object recognition memory were assessed during adulthood. Fear extinction learning was exclusively facilitated in the WP-AGIQ rats. Synaptic plasticity-related genes showed a similar pattern of constitutive expression changes in the hippocampal dentate gyrus and prelimbic medial prefrontal cortex (mPFC) between the DEV-AGIQ and WP-AGIQ rats. However, WP-AGIQ rats revealed more genes constitutively upregulated in the infralimbic mPFC and amygdala than DEV-AGIQ rats, as well as FOS-immunoreactive(+) neurons constitutively increased in the infralimbic cortex. Ninety minutes after the last fear extinction trial, many synaptic plasticity-related genes (encoding Ephs/Ephrins, glutamate receptors/transporters, and immediate-early gene proteins and their regulator, extracellular signal-regulated kinase 2 [ERK2]) were upregulated in the dentate gyrus and amygdala in WP-AGIQ rats. Additionally, WP-AGIQ rats exhibited increased phosphorylated ERK1/2+ neurons in both the prelimbic and infralimbic cortices. These results suggest that AGIQ exposure from the fetal stage to adulthood is necessary for facilitating fear extinction learning. Furthermore, constitutive and learning-dependent upregulation of synaptic plasticity-related genes/molecules may be differentially involved in brain regions that regulate fear memory. Thus, new learning-related neural circuits for facilitating fear extinction can be established in the mPFC.

Developmental exposure to diacetoxyscirpenol reversibly disrupts hippocampal neurogenesis by inducing oxidative cellular injury and suppressed differentiation of granule cell lineages in mice.

To investigate the developmental exposure effect of diacetoxyscirpenol (DAS) on postnatal hippocampal neurogenesis, pregnant ICR mice were provided a diet containing DAS at 0, 0.6, 2.0, or 6.0 ppm from gestational day 6 to day 21 on weaning after delivery. Offspring were maintained through postnatal day (PND) 77 without DAS exposure. On PND 21, neural stem cells (NSCs) and all subpopulations of proliferating progenitor cells were suggested to decrease in number in the subgranular zone (SGZ) at ≥ 2.0 ppm. At 6.0 ppm, increases of SGZ cells showing TUNEL+, metallothionein-I/II+, γ-H2AX+ or malondialdehyde+, and transcript downregulation of Ogg1, Parp1 and Kit without changing the level of double-stranded DNA break-related genes were observed in the dentate gyrus. This suggested induction of oxidative DNA damage of NSCs and early-stage progenitor cells, which led to their apoptosis. Cdkn2a, Rb1 and Trp53 downregulated transcripts, which suggested an increased vulnerability to DNA damage. Hilar PVALB+ GABAergic interneurons decreased and Grin2a and Chrna7 were downregulated, which suggested suppression of type-2-progenitor cell differentiation. On PND 77, hilar RELN+ interneurons increased at ≥ 2.0 ppm; at 6.0 ppm, RELN-related Itsn1 transcripts were upregulated and ARC+ granule cells decreased. Increased RELN signals may ameliorate the response to the decreases of NSCs and ARC-mediated synaptic plasticity. These results suggest that DAS reversibly disrupts hippocampal neurogenesis by inducing oxidative cellular injury and suppressed differentiation of granule cell lineages. The no-observed-adverse-effect level of DAS for offspring neurogenesis was determined to be 0.6 ppm (0.09-0.29 mg/kg body weight/day).

Effects of compound X, a novel potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase, on the adrenal gland of rats.

To investigate the adrenal toxicity of a novel inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase, compound X (CX), histopathological examinations, fat staining, adrenal cholesterol measurement, blood biochemistry, plasma corticosterone and ACTH measurement, ACTH-stimulation assay, and adrenal gene-expression analyses were done in rats in repeated-dose studies (experiment 1: 0, 3, 10, 30 and 150mg/kg for 4, 8, 15 and 28 days; experiment 2: 0, 3, 10,30 and 150mg/kg for 28 days; experiment 3: 0, 10, 30, 100 and 300mg/kg for 28 days). CX induced morphologic changes such as vacuolation and hypertrophy in the zona fasciculata (ZF) at ≥10mg/kg, and eosinophilic changes in the ZF at 150mg/kg. Vacuolation decreased in a dose-dependent manner and was replaced by eosinophilic changes. Inflammatory and fibrous changes were observed at ≥30mg/kg. These changes were expressed at early stages of dosing and were not exacerbated by extension of the administration period. Oil-red-O/Filipin staining showed depletion of cholesterol ester in dose-dependent manner and enabled adrenal cholesterol measurement. Filipin staining also revealed vacuoles to be composed of cholesterol esters. No significant changes were observed during the dosing period of CX for plasma corticosterone and ACTH levels. Gene-expression analyses showed up-regulation of Star and Abca1 mRNA levels at 300mg/kg. In conclusion, CX induced adrenal toxicity, but CX did not influence adrenocortical functions, and exacerbation of adrenal toxicities by extension of the administration period was not observed. Up-regulation of genes related to the transport of FC, such as Star and Abca1, were observed in CX groups, and these genes may be involved in the maintenance of adrenal structure and function in rats given CX.