• Animal cells and systems
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• v.12 no.4
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• pp.211-217
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• 2008

Bax, a pro-apoptotic member of Bcl-2 family proteins, plays a central role in the mitochondria-dependent apoptosis. Apoptotic signals induce the translocation of Bax from cytosol into the mitochondria, which triggers the release of apoptogenic molecules such as cytochrome C and apoptosis-inducing factor, AIF. Bax-inhibiting peptide(BIP) is a cell permeable peptide comprised of five amino acids designed from the Bax-interaction domain of Ku70. Because BIP inhibits Bax translocation and Bax-mediated release of cytochrome C, BIP suppresses Bax-dependent apoptosis. In this study, we observed that BIP inhibited staurosporine-induced neuronal death in cultured cerebral cortex and cerebellar granule cells, but BIP failed to rescue granule cells from trophic signal deprivation-induced neuronal death, although both staurosporine-induced and trophic signal deprivation-induced neuronal death are dependent on Bax. These findings suggest that the mechanisms of the Bax activation may differ depending on the type of cell death induction, and thus BIP exhibits selective suppression of a subtype of Bax-dependent neuronal death.

• Journal of Life Science
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• v.15 no.4 s.71
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• pp.607-612
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• 2005

Heat shock protein 70 (HSP70) is a multigene family composed of constitutively expressed members(Hsc70) and stress-inducible members (Hsp70). In the mammalian nervous system, a considerable amount of HSPs is also synthesized under normal conditions suggesting that they play an important role in the metabolism of unstressed cells. In this study we examined the expression of Hsp70 in the synapses of rat cerebellar neurons. Immunohistochemistry using specific antibodies revealed that both Hsp70 and Hsc70 are expressed in the cerebellar tissue, with strongest expression in Purkinje cells followed by granule cells. Neurons in deep cerebellar nuclei were also intensely stained by Hsp70 antibody. Immunocytochemical stainings of cultured cerebellar cells showed that Hsp70 is expressed in both Purkinje and granule cells. The expression was punctate in the soma and along dendritic trees, and the punctae were colocalized with those of PSD95, a postsynaptic marker. Immunoblotting also indicates that Hsp70 is associated with the postsynaptic density fraction. Taken together, our results indicate that the Hsp70 is expressed in cerebellar neurons in normal conditions, and that some are localized in the synapses.

• Animal cells and systems
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• v.1 no.4
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• pp.589-594
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• 1997

The neuronal voltage-sensitive calcium channels (VSCCs) are multisubunit complexes consisting of $\alpha_1,\;\alpha_2-\delta$ and $\beta$ subunits. Heterologous expression and biochemical studies have shown that the activity of VSCCs is regulated by their $\beta$ subunits in a $\beta$ subunit isoform-specific manner. To elucidate the $\beta$ subunit identity of the P/Q-type calcium channel encoded by an $\alpha_{1A}$ subunit, which is exclusively expressed in the Purkinje and granule cell of the cerebellum, we have examined the spatial and temporal expression patterns of $\beta$ subunits and compared them with those of $\alpha_{1A}$ subunit in the developing rat cerebellum. Reverse transcriptase- polymerase chain reaction (RT-PCR) and Northern blot analysis have shown that $\beta_4$ subunit mRNA was prominently expressed in the cerebellum and much more abundant than any other distinct $\beta$ subunits. RNase protection assay has further demonstrated that the expression of $\alpha_{1A}$ and $\beta_4$ subunits increased during cerebellar development, while the amount of $\beta_2$ and $\beta_3$ mRNAs did not significantly change. In addition, a $\beta_4$ transcript was present in cultured cerebellar granule cells, but not in astrocyte cells, and the level of $\beta_4$ mRNA expression increased gradually in vitro seen as in vivo. Based on the spatial and temporal expression patterns of $\beta_4$ subunit, we conclude that $\beta_4$ may predominantly associate, but probably not exclusively, with the $\alpha_{1A}$ subunit in rat cerebellar granule cells.

• Natural Product Sciences
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• v.10 no.6
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• pp.289-295
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• 2004

Myristica fragrans seed from Myristica fragrans Houtt (Myristicaceae) has various pharmacological activities peripherally and centrally. The present study aims to investigate the effect of the methanol extract of Myristica fragrans seed (MF) on N-methyl-D-aspartate (NMDA)-induced neurotoxicity in primary cultured rat cerebellar granule neuron. MF, over a concentration range of 0.05 to $5\;{\mu}g/ml$, inhibited NMDA (1 mM)- induced neuronal cell death, which was measured by trypan blue exclusion test and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. MF $(0.5\;{\mu}g/ml)$ inhibited glutamate release into medium induced by NMDA (1 mM), which was measured by HPLC. Pretreatrnent of MF $(0.5\;{\mu}g/ml)$ inhibited NMDA (1 mM)-induced elevation of cytosolic calcium concentration $([Ca^{2+}]_c)$, which was measured by a fluorescent dye, Fura 2-AM, and generation of reactive oxygen species (ROS). These results suggest that MF prevents NMDA-induced neuronal cell damage in vitro.

• Archives of Pharmacal Research
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• v.20 no.1
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• pp.7-12
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• 1997

Glutamate uptake inhibitor, L-trans-pyrrolidine-2, 4-dicarboxylate (PDC, $20{\mu}M$) elevated basal and N-methyl-D-aspartate (NMDA, $100{\mu}M$)-induced extracellular glutamate accumulation, while it did not augment kainate $100{\mu}M$-induced glutamate accumulation in cultured cerebellar granule neurons. However, pretreatment with PDC for 1 h significantly reduced NMDA-induced glutamate accumulation, but did not affect kainate-induced response. Pretreatment with glutamate $(5{\mu}M)$ for 1 h also reduced NMDA-induced glutamate accumulation, but did not kainate-induced response. Upon a brief application (3-10 min), PDC did neither induce elevation of intracellular calcium concentration $([Ca^{2+}]_i)$ nor modulate NMDA-indLiced $[Ca^{2+}]_1$ elevation. Pretreatment with PDC for 1 h reduced NMDA-induced $[Ca^{2+}]_1$ elevation, but it did not reduce kainate-induced $[Ca^{2+}]_1$ elevation. These results suggest that glutamate concentration in synaptic clefts of neurana cells is increased by prolonged exposure (1 h) of the cells to PDC, and the accumulated glutamate subsequently induces selective desensitization of NMDA receptor.

• Journal of Physiology & Pathology in Korean Medicine
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• v.27 no.6
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• pp.759-763
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• 2013

This experiment was performed to investigate whether 50% ethanol extracts of the combined-preparation of Longanae Arilus, Chrysanthemi Flos, Zizyphi Fructus and Ginseng Radix alba (CPE) has hypnotic effects and/or enhances pentobarbital-induced sleeping time. Locomotor activity was evaluated using a ambulometer of tilting-type. The sedative-hypnotic effects were evaluated by measuring the sleeping onset time and sleeping time in pentobarbital-treated mice 30 min. after oral administration of CPE and muscimol. The intracellular $Cl^-$ concentration of cerebellar granule cells was estimated using $Cl^-$ sensitive fluorescence probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium (MQAE). CPE (150 mg/kg) decreased the locomotor activity, but CPE itself did not induce sleep. However, CPE reduced sleeping onset and prolonged sleeping time induced by pentobarbital (42 mg/kg). In addition, CPE (2 ${\mu}g/ml$) and pentobarbital (2.5 ${\mu}M$) itself did not affect on the chloride influx in primary cultured cerebellar granule cells, but the combination of CPE and pentobarbital (2.5 ${\mu}M$) increased the chloride influx onto the cells. In conclusion, it is suggested that CPE might augment pentobarbital-induced sleep through the increase of chloride influx.

• Biomolecules & Therapeutics
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• v.16 no.3
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• pp.261-269
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• 2008

This experiment was performed to investigate the anxiolytic-like effects of cyclopeptide fraction alkaloids of Zizyphi Spinosi Semen (CFAZ), by using the experimental paradigms of anxiety, and compared with those of a known anxiolytic, diazepam. CFAZ (8.0 mg/kg, p.o.) increased the percentage of time spent on the open arms and the number of open arms entries in the elevated plus-maze test, increased the number of head dips in the hole-board test, and increased the percentage of center zone ambulatory time in the open-field box. However, CFAZ has no effect on the locomotor activity, while diazepam (2.0 mg/kg, p.o.) significantly reduced locomotor activity. CFAZ did not influence the grip force in the grip strength meter test, either. From the molecular experiments, CFAZ increased chloride influx in cultured cerebellar granule cells. In addition, $GABA_A$ receptors $\gamma$-subunit were over-expressed by CFAZ in cultured cerebellar granule cells. It is concluded that CFAZ may have anxiolytic-like effects, and these effects may be mediated by $GABA_A$ receptors.

• Korean Journal of Medicinal Crop Science
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• v.12 no.5
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• pp.415-423
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• 2004

Myristica fragrans seed from Myristica fragrans Houtt (Myristicaceae) has various pharmacological activities peripherally and centrally. The present study aims to investigate the effect of the methanol extract of Myristica fragrans seed (MF) on kainic acid (KA)-induced neurotoxicity in primary cultured rat cerebellar granule neuron. MF, over a concentration range of 0.05 to $5\;{\mu}g/ml$ inhibited KA $(500\;{\mu}M)-induced$ neuronal cell death, which was measured by trypan blue exclusion test and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. MF $(0.5\;{mu}g/ml)$ inhibited glutamate release into medium induced by KA $(500\;{\mu}M)$, which was measured by HPLC. Pretreatment of MF $(0.5\;{mu}g/ml)$ inhibited KA $(500\;{\mu}M)-induced$ elevation of cytosolic calcium concentration $([Ca^{2+}]_c)$, which was measured by a fluorescent dye, Fura 2-AM, and generation of reactive oxygen species (ROS). These results suggest that MF prevents KA-induced neuronal cell damage in vitro.

• The Korean Journal of Pharmacology
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• v.32 no.1
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• pp.13-21
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• 1996

Intracellular calcium concentration ($[Ca^{2+}]_i$) may play a crucial role in a variety of neuronal functions. Here we report that in primary culture of mouse cerebellar granule cells nicotinic acetylcholine receptors (nAChRs) are expressed in a specific developmental stage and involved in the regulation of intracellular calcium homeostasis. Nicotine-mediated calcium responses were measured using $^{45}Ca^{2+}$ or fluorometrically using the calcium-sensitive fluorescent dye fura-2. Maximal uptake of $^{45}Ca^{2+}$ evoked by nicotine in mouse cerebellar granule cells were revealed $8{\sim}12$ days in culture. In contrast, nicotine did not alter the basal $^{45}Ca^{2+}$ uptake in cultured glial cells. In cerebellar granule cells nicotine-evoked $^{45}Ca^{2+}$ uptake was largely blocked by the NMDA receptor antagonists. Glutamate pyruvate transaminase (GPT). which removes endogenous glutamate, also prevented nicotine effects, implying the indirect involvement of glutamate in nicotine-mediated calcium responses. Fluorometric studies using fura-2 showed two phases of nicotine-evoked $[Ca^{2+}]_i$ rises: the initial rising phase and the later plateau phase. Interestingly, the NMDA receptor antagonists and GPT appeared to inhibit only the later plateau phase of nicotine-evoked $[Ca^{2+}]_i$ rises. The present results imply that nicotine mediated $^{45}Ca^{2+}$ uptake and $[Ca^{2+}]_i$ rises are attributed to the calcium fluxes through both nAchRs and NMDA receptors in a time-dependent manner. Consequently, nAChRs may play an important role in neuronal development by being expressed in a specific developmental stage and regulating the intracellular calcium homeostasis.

• Biomolecules & Therapeutics
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• v.16 no.4
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• pp.328-335
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• 2008

This study was undertaken to investigate whether honokiol could enhance the pentobarbitalinduced sleeping behaviors through $\gamma$-aminobutyric acid (GABA) receptor $Cl^-$ channel activation. Thirty minutes after the oral administration of honokiol, mice were received sodium pentobarbital (42 mg/kg, i.p.). The time elapsed from pentobarbital injection to the loss of the righting reflex was taken as sleeping latency. The time elapsed between the loss and voluntary recovery of the righting reflex was considered as the total sleeping time. Western blot technique and $Cl^-$ sensitive fluorescence probe were used to detect the expression of $GABA_A$ receptor subunits and $Cl^-$ influx in the primary cultured cerebellar granule cells. Honokiol (0.1 and 0.2 mg/kg) prolonged the sleeping time induced by pentobarbital (42 mg/kg) in a dosage-dependent manner. Honokiol (20 and 50 ${\mu}M$) increased $Cl^-$ influx in primary cultured cerebellar granule cells, and selectively increased the $GABA_A$ receptor $\alpha$-subunit expression, but had no effect on the abundance of $\beta$ or $\gamma$-subunits. Chronic treatment with 20 ${\mu}M$ honokiol in primary cultured cerebellar neurons did not affect the abundance of GAD65/67. The results suggested that honokiol could potentiate pentobarbital-induced sleeping through $GABA_A$ receptor $Cl^-$ channel activation.