FOXG1 Directly Suppresses Wnt5a During the Development of the Hippocampus / 神经科学通报·英文版

The Wnt signaling pathway plays key roles in various developmental processes. Wnt5a, which activates the non-canonical pathway, has been shown to be particularly important for axon guidance and outgrowth as well as dendrite morphogenesis. However, the mechanism underlying the regulation of Wnt5a remains unclear. Here, through conditional disruption of Foxg1 in hippocampal progenitors and postmitotic neurons achieved by crossing Foxg1

The Joubert Syndrome Gene arl13b is Critical for Early Cerebellar Development in Zebrafish / 神经科学通报·英文版

Joubert syndrome is characterized by unique malformation of the cerebellar vermis. More than thirty Joubert syndrome genes have been identified, including ARL13B. However, its role in cerebellar development remains unexplored. We found that knockdown or knockout of arl13b impaired balance and locomotion in zebrafish larvae. Granule cells were selectively reduced in the corpus cerebelli, a structure homologous to the mammalian vermis. Purkinje cell progenitors were also selectively disturbed dorsomedially. The expression of atoh1 and ptf1, proneural genes of granule and Purkinje cells, respectively, were selectively down-regulated along the dorsal midline of the cerebellum. Moreover, wnt1, which is transiently expressed early in cerebellar development, was selectively reduced. Intriguingly, activating Wnt signaling partially rescued the granule cell defects in arl13b mutants. These findings suggested that Arl13b is necessary for the early development of cerebellar granule and Purkinje cells. The arl13b-deficient zebrafish can serve as a model organism for studying Joubert syndrome.

The Joubert Syndrome Gene arl13b is Critical for Early Cerebellar Development in Zebrafish / 神经科学通报·英文版

Joubert syndrome is characterized by unique malformation of the cerebellar vermis. More than thirty Joubert syndrome genes have been identified, including ARL13B. However, its role in cerebellar development remains unexplored. We found that knockdown or knockout of arl13b impaired balance and locomotion in zebrafish larvae. Granule cells were selectively reduced in the corpus cerebelli, a structure homologous to the mammalian vermis. Purkinje cell progenitors were also selectively disturbed dorsomedially. The expression of atoh1 and ptf1, proneural genes of granule and Purkinje cells, respectively, were selectively down-regulated along the dorsal midline of the cerebellum. Moreover, wnt1, which is transiently expressed early in cerebellar development, was selectively reduced. Intriguingly, activating Wnt signaling partially rescued the granule cell defects in arl13b mutants. These findings suggested that Arl13b is necessary for the early development of cerebellar granule and Purkinje cells. The arl13b-deficient zebrafish can serve as a model organism for studying Joubert syndrome.

Morin Prevents Granule Cell Dispersion and Neurotoxicity via Suppression of mTORC1 in a Kainic Acid-induced Seizure Model

An abnormal reorganization of the dentate gyrus and neurotoxic events are important phenotypes in the hippocampus of patients with temporal lobe epilepsy (TLE). The effects of morin, a bioflavonoid constituent of many herbs and fruits, on epileptic seizures have not yet been elucidated, though its beneficial effects, such as its anti-inflammatory and neuroprotective properties, are well-described in various neurodegenerative diseases. In the present study, we investigated whether treatment with morin hydrate (MH) can reduce the susceptibility to seizures, granule cell dispersion (GCD), mammalian target of rapamycin complex 1 (mTORC1) activity, and the increases in the levels of apoptotic molecules and inflammatory cytokines in the kainic acid (KA)-induced seizure mouse model. Our results showed that oral administration of MH could reduce susceptibility to seizures and lead to the inhibition of GCD and mTORC1 activity in the KA-treated hippocampus. Moreover, treatment with MH significantly reduced the increased levels of apoptotic signaling molecules and pro-inflammatory mediators in the KA-treated hippocampus compared with control mice, suggesting a neuroprotective role. Therefore, these results suggest that morin has a therapeutic potential against epilepsy through its abilities to inhibit GCD and neurotoxic events in the in vivo hippocampus.

Developmental Exposure to Di-(2-ethylhexyl) Phthalate Induces Cerebellar Granule Cell Apoptosis via the PI3K/AKT Signaling Pathway

Di-(2-ethylhexyl) phthalate (DEHP) is an ubiquitous environmental contaminant because of its extensive use in plastics and its persistence. As an environmental endocrine disruptor, it is suspected to interfere with neurodevelopment in people. However, evidence of the effects of maternal DEHP exposure on cerebellar development in offspring is scarce. The objective of this study was to investigate maternal exposure to DEHP and its effect on apoptosis of cerebellar granule cells (CGCs) and related mechanisms. Pregnant Wistar rats were administrated DEHP (0, 30, 300 and 750 mg/kg/d) by gavage from gestational day (GD) 0 to postnatal day (PN) 21. Primary CGCs were also exposed to mono-(2-ethylhexyl) phthalate (MEHP), the main metabolite of DEHP, for 24 h with concentrations of 0, 25, 100 and 250 µM. The CGCs of male offspring from 300 and 750 mg/kg/d DEHP exposure groups showed significantly increased apoptosis. In addition, the PI3K/AKT signaling pathway was inhibited in the male offspring of the 300 and 750 mg/kg/d DEHP exposure groups. However, effects on female pups were not obvious. Apoptosis was also elevated and the PI3K/AKT signaling pathway was inhibited after primary CGCs were exposed to MEHP. Furthermore, apoptosis was reduced after treatment with the PI3K/AKT signaling pathway activator, insulin-like growth factor (IGF) 1, and increased after treatment with LY294002, an inhibitor of the PI3K/AKT signaling pathway. These results suggested that maternal DEHP exposure induced apoptosis in the CGCs of male pups via the PI3K/AKT signaling pathway, and the apoptosis could be rescued by IGF1 and aggravated by LY294002.

Beneficial Effects of Silibinin Against Kainic Acid-induced Neurotoxicity in the Hippocampus in vivo

Silibinin, an active constituent of silymarin extracted from milk thistle, has been previously reported to confer protection to the adult brain against neurodegeneration. However, its effects against epileptic seizures have not been examined yet. In order to investigate the effects of silibinin against epileptic seizures, we used a relevant mouse model in which seizures are manifested as status epilepticus, induced by kainic acid (KA) treatment. Silibinin was injected intraperitoneally, starting 1 day before an intrahippocampal KA injection and continued daily until analysis of each experiment. Our results indicated that silibinin-treatment could reduce seizure susceptibility and frequency of spontaneous recurrent seizures (SRS) induced by KA administration, and attenuate granule cell dispersion (GCD), a morphological alteration characteristic of the dentate gyrus (DG) in temporal lobe epilepsy (TLE). Moreover, its treatment significantly reduced the aberrant levels of apoptotic, autophagic and pro-inflammatory molecules induced by KA administration, resulting in neuroprotection in the hippocampus. Thus, these results suggest that silibinin may be a beneficial natural compound for preventing epileptic events.

Preventive effects of imperatorin on perfluorohexanesulfonate-induced neuronal apoptosis via inhibition of intracellular calcium-mediated ERK pathway

Early life neuronal exposure to environmental toxicants has been suggested to be an important etiology of neurodegenerative disease development. Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds, is widely distributed environmental contaminants. We have reported that PFHxS induces neuronal apoptosis via ERK-mediated pathway. Imperatorin is a furanocoumarin found in various edible plants and has a wide range of pharmacological effects including neuroprotection. In this study, the effects of imperatorin on PFHxS-induced neuronal apoptosis and the underlying mechanisms are examined using cerebellar granule cells (CGC). CGC were isolated from seven-day old rats and were grown in culture for seven days. Caspase-3 activity and TUNEL staining were used to determine neuronal apoptosis. PFHxS-induced apoptosis of CGC was significantly reduced by imperatorin and PD98059, an ERK pathway inhibitor. PFHxS induced a persistent increase in intracellular calcium, which was significantly blocked by imperatorin, NMDA receptor antagonist, MK801 and the L-type voltage-dependent calcium channel blockers, diltiazem and nifedipine. The activation of caspase-3 by PFHxS was also inhibited by MK801, diltiazem and nifedipine. PFHxS-increased ERK activation was inhibited by imperatorin, MK801, diltiazem and nifedipine. Taken together, imperatorin protects CGC against PFHxS-induced apoptosis via inhibition of NMDA receptor/intracellular calcium-mediated ERK pathway.

Systemic administration of low dosage of tetanus toxin decreases cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus / 한국실험동물학회지

In the present study, we investigated the effect of Tetaus toxin (TeT) on cell proliferation and neuroblast differentiation using specific markers: 5-bromo-2-deoxyuridine (BrdU) as an exogenous marker for cell proliferation, Ki-67 as an endogenous marker for cell proliferation and doublecortin (DCX) as a marker for neuroblasts in the mouse hippocampal dentate gyrus (DG) after TeT treatment. Mice were intraperitoneally administered 2.5 and 10 ng/kg TeT and sacrificed 15 days after the treatment. In both the TeT-treated groups, no neuronal death occurred in any layers of the DG using neuronal nuclei (NeuN, a neuron nuclei maker) and Fluoro-Jade B (F-J B, a high-affinity fluorescent marker for the localization of neuronal degeneration). In addition, no significant change in glial activation in both the 2.5 and 10 ng/kg TeT-treated-groups was found by GFAP (a marker for astrocytes) and Iba-1 (a marker for microglia) immunohistochemistry. However, in the 2.5 ng/kg TeT-treated-group, the mean number of BrdU, Ki-67 and DCX immunoreactive cells, respectively, were apparently decreased compared to the control group, and the mean number of each in the 10 ng/kg TeT-treated-group was much more decreased. In addition, processes of DCX-immunoreactive cells, which projected into the molecular layer, were short compared to those in the control group. In brief, our present results show that low dosage (10 ng/kg) TeT treatment apparently decreased cell proliferation and neuroblast differentiation in the mouse hippocampal DG without distinct gliosis as well as any loss of adult neurons.

Chlorination of ortho-position on Polychlorinated Biphenyls Increases Protein Kinase C Activity in Neuronal Cells

Polychlorinated biphenyls (PCBs) are persistent and bioaccumulative environmental pollutants. Recently, it is suggested that neurotoxic effects such as motor dysfunction and impairment in memory and learning have been associated with PCB exposure. However, structure relationship of PCB congeners with neurotoxic effects remains unknown. Since PKC signaling pathway is implicated in the modulation of motor behavior as well as learning and memory and the role of PKC are subspecies-specific, we attempted to study the effects of structurally distinct PCBs on the total PKC activity as well as subspecies of PKC in cerebellar granule cell culture model. Cells were exposed to 0, 25 and 50 microM of PCB-126, PCB-169, PCB-114, PCB-157, PCB-52 and PCB-4 for 15 min. Cells were subsequently analyzed by [3H] phorbol ester binding assay or immunoblotted against PKC-alpha and -epsilon monoclonal antibodies. While non-dioxin-like-PCB (PCB-52 and PCB-4) induced a translocation of PKC-alpha and -epsilon from cytosol to membrane fraction, dioxin-like PCBs (PCB-126, -169, -114, -157) had no effects. [3H] Phorbol ester binding assay also revealed structure-dependent increase similar to translocation of PKC isozymes. While PCB-4 induced translocation of PKC-alpha and -epsilon was inhibited by ROS inhibitor, the pattern of translocation was not affected in presence of AhR inhibitor. It is suggested that PCB-4-induced PKC activity may not be mediated via AhR-dependent pathway. Taken together, our findings suggest that chlorination of ortho-position in PCB may be a critical structural moiety associated with neurotoxic effects, which may be preferentially mediated via non-AhR-dependent pathway. Therefore, the present study may contribute to understanding the neurotoxic mechanism of PCBs as well as providing a basis for establishing a better neurotoxic assessment.

The Effect of the Gekko Gecko Ethanol Extract on the Granular Cells Apoptosis of Rat Ovaries / 实用妇产科杂志

Objective:To study the effects of the Gekko gecko ethanol extract on granular cells apoptosis of rat ovaries.Methods :3 month and 6 month female Wistar rats were taken in this study, and 20 rats were in each age group.Each age group were randomly divided into two groups(experimental group and control tragastric administration in experimental group, corresponding volume of 0.9% sodium chloride was given in control group by the same way.Apoptosis of granular cells and distribution of cell cycle in rat ovary were measured by terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling (TUNEL) and propidi-um iodide(PI) staining, AnnexinV/PI double labelling staining by flow cytometry (FCM) after administration for 15 days.Re8utts:The apoptosis numbers of granular cells in 3 month and 6 month age experimental group were 34 ±7 and 53 ±11, in control group were 48±9 and 76±17 respectively by TUNEL.There was signrficantly statistic difference (P <0.01).The apoptosis rates in 3 month and 6 month age experimental group were(6.06 ±2.01)% and (12.16 ±3.26) % , in the control group were (8.23±2.13) % and (23.69 ±6.28)% respectively by PI staining.There was significantly statistic drfference (P <0.05).However, no significant difference was found between two groups in cellular cycle distribution (P > 0.05).Early apoptosis rates in 3 month and 6 month age experimental group were (6.71±3.11) % and (23.74±6.28) % , in the control group were (19.05 ±5.78) % and (36.55± 8.35) % respectively by AnnexinV/PI double labelling staining FCM.There was significantly statistic difference (P <0.01).The apoptosis of granular cells were sig-nificantly less in experimental groups comparing wrth control groups both in 3 month and 6 month age group (P<0.01 or P<0.05).Conclusions :The ethanol extract of gekko gecko can inhibit the apoptosis of granu-lar cells in rat ovary, then improves the ovary function and delay ageing of rat.

Activity of Single Granule Cell in the Dentate Gyrus of Guinea Pig During The Consolidation of Trace Eyeblink Conditioned Responses / 生物化学与生物物理进展

The hippocampus plays a critical role during the consolidation of trace eyeblink conditioned responses (CRs). However, the role of its related structure such as dentate gyms (DG) remains unclear. The present study was aimed at monitoring the activity of single granule cell in the DG during the consolidation of trace eyeblink CRs, and elucidating the possible role of DG during this hippocampus-dependent task. Guinea pigs (n=8) were trained on a trace eyeblink conditioning paradigm using a 200-ms tone conditioned stimulus (CS), a 200-ms corneal airpuff unconditioned stimulus (US) and a 600-ms trace interval. Controls consisted of pseudo- conditioned guinea pigs (n=8). Extracellular single unit recordings in vivo were performed in the DG of learner animals during the consolidation of trace eyeblink CRs. The results revealed that all the trace-conditioned animals acquired the trace eyeblink CRs over 14 training days, however, none of the pseudo-conditioned animals did. Furthermore, 23 of 40 single granule cells in the DG of learner animals exhibited heterogeneous activity patterns during the consolidation of trace eyeblink CRs such as increases in activities to the tone CS, trace interval or airpuff US. The results suggested that the DG might participate in the neural circuit important for the consolidation of trace eyeblink CRs, and that the granule cells might encode different information during the consolidation of trace eyeblink CRs.

Role of cGMP-dependent protein kinase against ethanol neurotoxicity of cerebellar granule cells / 第三军医大学学报

Objective To analyze the function of cGMP-dependent protein kinase(PKG)in the survival of cultured rat cerebellar granule cells(CGC).Methods CGC culture was incubated for 24 h before addition of ethanol and different reagents,such as Deta-NONOate(a NO donor),Br-cGMP and Rp-8-pCPT-cGMPS(a cGMP-dependent protein kinase inhibitor).After the treatment,samples were incubated for another 24 h,then the cells were collected and counted by using hemocytometer.Results Deta-NONOate and Br-cGMP enhanced the cell survival in the absence of ethanol,indicating their neurotrophic effects.They showed neuroprotective effect on the cultured CGC since they could reduce the ethanol-induced cell death.Inhibiting PKG with Rp-8-pCPT-cGMPS could eliminate both neurotrophic and neuroprotective effects mediated by Deta-NONOate and Br-cGMP.Conclusion cGMP-dependent protein kinase plays a key role in the cultured CGC survival and protects the cells against ethanol neurotoxicity.

Calcium Influx is Responsible for Afterdepolarizations in Rat Hippocampal Dentate Granule Cells

Granule cells in dentate gyrus of hippocampus relay information from entorhinal cortex via perforant fiber to pyramidal cells in CA3 region. Their electrical activities are known to be closely associated with seizure activity as well as memory acquisition. Since action potential is a stereotypic phenomena which is based on all-or-none principle of Na+ current, the neuronal firing pattern is mostly dependent on afterpotentials which follows the stereotypic Na+ spike. Granule cells in dentate gyrus show afterdepolarization (ADP), while interneurons in dentate gyrus have afterhyperpolarizaton. In the present study, we investigated the ionic mechanism of afterdepolarization in hippocampal dentate granule cell. Action potential of dentate granule cells showed afterdepolarization, which was characterized by a sharp notch followed by a depolarizing hump starting at about -49.04 +/- 1.69 mV (n=43, mean +/- SD) and lasting 3~7 ms. Increase of extracellular Ca2+ from 2 mM to 10 mM significantly enhanced the ADP both in amplitude and in duration. A K+ channel blocker, 4-aminopyridine (4-AP, 2 mM), enhanced the ADP and often induced burst firings. These effects of 10 mM Ca2+ and 4-AP were additive. On the contrary, the ADP was significantly suppressed by removal of external Ca2+, even in the presence of 4-AP (2 mM). A Na+ channel blocker, TTX (100 nM), did not affect the ADP. From these results, it is concluded that the extracellular Ca2+ influx contributes to the generation of ADP in granule cells.

Distribution and synaptic organization of nitric oxide synthase immunoreactive neurons in the rat olfactory / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Nitric oxide (NO) has been reported to play important roles in the regulation of olfactory information in the mammarian olfactory bulb. Although the distribution of nitric oxide synthase (NOS)-immunoreactive neurons in the olfactory bulb in the rat and other animals have been investigated by light microscopy, ultrastructures of the synaptic organization between NOS-immunoreactive neurons have not been studied yet. This study was conducted in order to identify NOS- immunoreactive neurons in the rat olfactory bulb and to define their synaptic organizations under the electron microscope using the preembedding immunocytochemical method which utilizes anti-NOS antiserum. MATERIALS AND METHODS: The olfactory bulbs of the rats were cut into 50 micromiter thick vertical sections and immunostained using the ABS method. Stained sections were observed under the light microscope. Some of the stained sections, additionally stained with uranyl acetate and dehydrated, were embedded in Epon 812 and prepared into 80 nm thick sections to be observed under the electron microscope. RESULT: NOS-immunoreactive neurons of the rat olfactory bulb made up 25.0% of periglomerular cells and 18.9% of granule cells. NOS-immunoreactive periglomerular cells received synaptic input from unlabeled axon terminals of the olfactory nerve and unlabeled periglomerular cells within the glomeruli. The output targets of NOS immunoreactive periglomerular cells were unlabeled axon terminals of the olfactory nerve and unlabeled periglomerular cells. NOS-immunoreactive granule cells received synaptic input from unlabeled processes of granule cells and axon terminals of mitral cells, and made output synapses onto the unlabeled axon terminals of mitral cells. CONCLUSION: NOS-immunoreactive neurons are periglomerular cells and granule cells, and NO liberated from NOS cells may play important roles in the modulation of olfactory transmission.

Differential effect of homocysteic acid and cysteic acid on changes of inositol phosphates and (Ca2+)i in rat cerebellar granule cells

The present study was undertaken to characterize homocysteic acid (HCA)-and cysteic acid (CA)mediated formation of inositol phosphates (InsP) in primary culture of rat cerebellar granule cells. HCA and CA stimulated InsP formation in a dose-dependent manner, which was prevented by the N-methyl-D-aspartate (NMDA) receptor antagonist D,L-2-amino-5-phosphopentanoic acid (APV). CA-, but not HCA-, mediated InsP formation was in part prevented by the metabotropic glutamate receptor antagonist alpha-methyl-4-carboxyphenylglycine ((+/-)-MCPG). Both HCA- and CA-mediated increases in intracellular calcium concentration were completely blocked by APV, but were not altered by (+/-)-MCPG. CA-mediated InsP formation was in part prevented by removal of endogenous glutamate. In contrast, the glutamate transport blocker L-aspartic acid-beta-hydroxamate synergistically increased CA responses. These data indicate that in cerebellar granule cells HCA mediates InsP formation wholly by activating NMDA receptor. In contrast, CA stimulates InsP formation by activating both NMDA receptor and metabotropic glutamate receptor, and in part by releasing endogenous glutamate into extracellular milieu.