• The Korean Journal of Physiology and Pharmacology
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• v.20 no.1
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• pp.101-109
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• 2016

Reducing $[Mg^{2+}]_o$ to 0.1 mM can evoke repetitive $[Ca^{2+}]_i$ spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM $[Mg^{2+}]_o$ are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether $Ca^{2+}$ spikes are involved in this process. Preconditioning by an exposure to 0.1 mM $[Mg^{2+}]_o$ for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type $Ca^{2+}$ channel antagonist nimodipine, which blocked 0.1 mM $[Mg^{2+}]_o$-induced $[Ca^{2+}]_i$ spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the $[Ca^{2+}]_i$ spikes. The intracellular $Ca^{2+}$ chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the $[Ca^{2+}]_i$ spikes. While $G{\ddot{o}}6976$, a specific inhibitor of $PKC{\alpha}$ had no effect on the tolerance, both the $PKC{\varepsilon}$ translocation inhibitor and the $PKC{\zeta}$ pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the $[Ca^{2+}]_i$ spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low $[Mg^{2+}]_o$ preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the $[Ca^{2+}]_i$ spike-induced activation of $PKC{\varepsilon}$ and $PKC{\xi}$, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.

• Biomolecules & Therapeutics
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• v.21 no.4
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• pp.299-306
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• 2013

In the present study, we investigated the effect of ethanolic extract of the seed of Zizyphus jujuba var. spinosa (EEZS) on cholinergic blockade-induced memory impairment in mice. Male ICR mice were treated with EEZS. The behavioral tests were conducted using the passive avoidance, the Y-maze, and the Morris water maze tasks. EEZS (100 or 200 mg/kg, p.o.) significantly ameliorated the scopolamine-induced cognitive impairment in our present behavioral tasks without changes of locomotor activity. The ameliorating effect of EEZS on scopolamine-induced memory impairment was significantly reversed by a sub-effective dose of MK-801 (0.0125 mg/kg, s.c.). In addition, single administration of EEZS in normal naive mouse enhanced latency time in the passive avoidance task. Western blot analysis was employed to confirm the mechanism of memory-ameliorating effect of EEZS. Administration of EEZS (200 mg/kg) increased the level of memory-related signaling molecules, including phosphorylation of extracellular signal-regulated kinase or cAMP response element-binding protein in the hippocampal region. Also, the time-dependent expression level of brain-derived neurotrophic factor by the administration of EEZS was markedly increased from 3 to 9 h. These results suggest that EEZS has memory-ameliorating effect on scopolamine-induced cognitive impairment, which is mediated by the enhancement of the cholinergic neurotransmitter system, in part, via NMDA receptor signaling, and that EEZS would be useful agent against cognitive dysfunction such as Alzheimer's disease.

• The Korean Journal of Pharmacology
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• v.32 no.2
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• pp.127-138
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• 1996

The effects of adenosine analogues on the electrically-evoked acetylcholine(ACh) release and the influence of ischemia on the effects were studied in the rat hippocampus. Slices from the rat hippocampus were equilibrated with $0.1{\mu}M$ $[^3H]-choline$ and the release of the labelled product, $[^3H]-ACh$, was evoked by electrical stimulation(3 Hz, 2 ms, 5 $VCm^{-1}$ and rectangular pulses for 2 min), and the influence of various agents on the evoked tritiumoutflow was investigated. Ischemia(10 min with 95% $N_2$ + 5% $CO_2$) increased both the basal and evoked ACh release. These increases were abolished by glucose addition into the superfused medium, and they significantly inhibited either by 0.1 & $0.3{\mu}M$ TTX pretreatment or by removing $Ca^{++}$ in the medium. MK-801($1{\sim}10{\mu}M$), a specific NMDA receptor antagonist, and glibenclamide $(1{\mu}M)$, a $K^+-channel$ inhibitor, did not alter the evoked ACh release and nor did they affect the ischemia-induced increases In ACh release. However, polymyxin B(0.03 mg), a specific protein kinase C inhibitor, significantly inhibited the effects of ischemia on the evoked ACh release. Adenosine and $N^6-cyclopentyladenosine$ decreased the ACh release in a dose dependent manner in ischemic condition, though the magnitude of inhibition was far less than those in normal(normoxic) condition. However, the treatment with $5{\mu}M$ DPCPX, a potent $A_1-adenosine$ receptor antagonist, potentiated the ischemia-effect. These results indicate that the evoked-ACh release is potentiated by ischemia, and this process being most probably mediated by protein kinase C, and that the decreased effect of ACh release mediated by $A_1-adenosine$ receptor is significantly inhibited in ischemic state.

• Archives of Pharmacal Research
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• v.28 no.3
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• pp.335-342
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• 2005

The effects of ginsenosides Rg$_3$(R) , Rg$_3$(S) and Rg$_5$/Rk$_1$ (a mixture of Rg$_5$ and Rk$_1$ 1:1, w/w), which are components isolated from processed Panax ginseng C.A. Meyer (Araliaceae), on memory dysfunction were examined in mice using a passive avoidance test. The ginsenosides Rg3(R), Rg3(S) or Rg$_5$/Rk$_1$, when orally administered for 4 days, significantly ameliorated the memory impairment induced by the single oral administration of ethanol. The memory impairment induced by the intraperitoneal injection of scopolamine was also significantly recovered by ginsenosides Rg3(S) and Rg$_5$/Rk$_1$. Among the three ginsenosides tested in this study, Rg$_5$/Rk$_1$ enhanced the memory function of mice most effectively in both the ethanol­and scopolamine-induced amnesia models. Moreover, the latency period of the Rg$_5$/Rk$_1$­treated mice was 1.2 times longer than that of the control (no amnesia) group in both models, implying that Rg$_5$/Rk$_1$ may also exert beneficial effects in the normal brain. We also evaluated the effects of these ginsenosides on the excitotoxic and oxidative stress-induced neuronal cell damage in primary cultured rat cortical cells. The excitotoxicity induced by glutamate or N­methyl-D-aspartate (NMDA) was dramatically inhibited by the three ginsenosides. Rg$_3$(S) and Rg$_5$/Rk$_1$ exhibited a more potent inhibition of excitotoxicity than did Rg$_3$(R). In contrast, these ginsenosides were all ineffective against the H$_2$O$_2$- or xanthine/xanthine oxidase-induced oxidative neuronal damage. Taken together, these results indicate that ginsenosides Rg$_3$(S) and Rg$_5$/Rk$_1$ significantly reversed the memory dysfunction induced by ethanol or scopolamine, and their neuroprotective actions against excitotoxicity may be attributed to their memory enhancing effects.

• The Korean Journal of Pharmacology
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• v.29 no.1
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• pp.9-22
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• 1993

To evaluate the protective effects of calcium antagonists, oxygen radical scavengers and excitatory amino acid (EAA) antagonist on the ischemic brain damage, we induced in vitro ischemic condition (namely, lack of oxygen and glucose) to rat hippocampal slices. And the degree of ischemic damage was determined by assaying changes in biochemical parameters such as ATP content and lactate ralease, MDA production in the presence or absence of the various drugs. During experimental ischemia for up to 60 min, ATP content was decreased and the amount of lactate release was markedly increased time-dependently. By changing the reaction medium which contained oxygen and glucose those biochemical parameters were recovered. But the recovery was not complete in this experimental condition. In the same ischemic conditions verapamil and vitamine E prevented the decrease of ATP content and the increase of lactate release from the slices. And verapamil and diltiazem decreased MDA release to the reaction medium. Superoxide dismutase (SOD) and MK-801 (as EAA receptor antagonist) protected the decrease of ATP content and reduced MDA release in 20 min ischemic condition, but glutathione affected ATP content and lactate release at the same condition. When oxygen and glucose were resupplied for 20 min after ischemic condition, verapamil showed the protective effect on the changes of ATP content and lactate release, and vitamine E decreased lactate release (at 20 min ischemia) and MDA release (at 60 min ischemia). These results showed that calcium antagonist and vitamine E protect the ischemic biochemical changes from rat hippocampal slices and calcium antagonist is more potent than vitamine E to protect the ischemical brain damege.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.5
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• pp.427-434
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• 2015

Significant evidence supports the role of the vestibular system in the regulation of blood pressure during postural movements. In the present study, the role of the vestibulo-spino-adrenal (VSA) axis in the modulation of blood pressure via the vestibulosympathetic reflex was clarified by immunohistochemical and enzyme immunoassay methods in conscious rats with sinoaortic denervation. Expression of c-Fos protein in the intermediolateral cell column of the middle thoracic spinal regions and blood epinephrine levels were investigated, following microinjection of glutamate receptor agonists or antagonists into the medial vestibular nucleus (MVN) and/or sodium nitroprusside (SNP)-induced hypotension. Both microinjection of glutamate receptor agonists (NMDA and AMPA) into the MVN or rostral ventrolateral medullary nucleus (RVLM) and SNP-induced hypotension led to increased number of c-Fos positive neurons in the intermediolateral cell column of the middle thoracic spinal regions and increased blood epinephrine levels. Pretreatment with microinjection of glutamate receptor antagonists (MK-801 and CNQX) into the MVN or RVLM prevented the increased number of c-Fos positive neurons resulting from SNP-induced hypotension, and reversed the increased blood epinephrine levels. These results indicate that the VSA axis may be a key component of the pathway used by the vestibulosympathetic reflex to maintain blood pressure during postural movements.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.3
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• pp.297-305
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• 1998

This study used in vivo intracellular recording in rat hippocampus to evaluate the effect of lidocaine and MK-801 on the membrane properties and the synaptic responses of individual neurons to electrical stimulation of the commissural pathway. Cells in control group typically fired in a tonic discharge mode with an average firing frequency of $2.4{\pm}0.9$ Hz. Neuron in MK-801 treated group (0.2 mg/kg, i.p.) had an average input resistance of $3.28{\pm}5.7\;M{\Omega}$ and a membrane time constant of $7.4{\pm}1.8$ ms. These neurons exhibited $2.4{\pm}0.2$ ms spike durations, which were similar to the average spike duration recorded in the neurons of the control group. Slightly less than half of these neurons were firing spontaneously with an average discharge rate of $2.4{\pm}1.1$ Hz. The average peak amplitude of the AHP following the spikes in these groups was $7.4{\pm}0.6$ mV with respect to the resting membrane potential. Cells in MK-801 and lidocaine treated group (5 mg/kg, i.c.v.) had an average input resistance of $3.45{\pm}6.0\;M{\Omega}$ and an average time constant of $8.0{\pm}1.4$ ms. The cells were firing spontaneously at an average discharge rate of $0.6{\pm}0.4$ Hz. Upon depolarization of the membrane by 0.8 nA for 400 ms, all of the tested cells exhibited accommodation of spike discharge. The most common synaptic response contained an EPSP followed by early-IPSP and late-IPSP. Analysis of the voltage dependence revealed that the early-IPSP and late-IPSP were putative $Cl^--and\;K^+-dependent$, respectively. Systemic injection of the NMDA receptor blocker, MK-801, did not block synaptic responses to the stimulation of the commissural pathway. No significant modifications of EPSP, early-IPSP, or late-IPSP components were detected in the MK-801 and/or lidocaine treated group. These results suggest that MK-801 and lidocaine manifest their CNS effects through firing pattern of hippocampal pyramidal cells and neural network pattern by changing the synaptic efficacy and cellular membrane properties.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2003.11a
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• pp.63-63
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• 2003

Opuntia ficus-indicavar. saboten Makino (Cactaceae) is a tropical or subtropical plant that has been widely used as folk medicine for the treatment of diabetes, asthma, burn, edema and gastritis. The purposes of the present study were to identify antioxidant constituents from fruits and stems of the plant cultivated in Cheju island, Korea, and examine their in vitro neuroprotective activities. Using a chromatographic fractionation method, ten chemical constituents were isolated from ethyl acetate extracts. By means of chemical and spectroscopic methods, those were identified as eight flavonoids such as kaempherol (a), quercetin (b), kaempferol 3-methyl ether (c), quercetin 3-methyl ether (d), narcissin (e), dihydrokaernpferol (f), dihydroquercetin (g) and erioclictyol (h), and two terpenoids such as 3-oxo-${\alpha}$-ionol-${\beta}$-d-glucopyranoside (i) and roseoside (j). Among the isolated compounds, comrounds c~e and h~j were those reported for the first titre from the plant. Compounds b, d and g showed DPPH free radical scavenging activities with IC$\sub$50/ values of 28, 19 and 31, ${\mu}$M respectively. Compounds d and g also inhibited iron-dependent lipid peroxidation with IC$\sub$50/ values of 2.4 and 3.5 ${\mu}$M. In a primary rat cortical neuronal cell culture system, compounds b, d and g inhibited xanthine/xanthine oxidase-induced (IC$\sub$50/ values of 18.2, 2.1 and 54.6 ${\mu}$M) and H$_2$O$_2$-induced (IC$\sub$50/ values of 13.6, 1.9 and 25.7 ${\mu}$M) cytotoxicities. In addition, compounds d and g inhibited NMDA-induced excitotoxicity by 21 and 33%, and only compound d inhibited growth factor withdrawal-induced apoptosis by 31% at a tested concentration of 3 ${\mu}$M. The results suggest that the antioxidant constituents with in vitroneuroprotective activities may serve as lead chemicals for the development of neuroprotective agent.

• Neonatal Medicine
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• v.17 no.2
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• pp.181-192
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• 2010

Purpose: Current studies have demonstrated the neuroprotective effects of dizocilpine (MK-801) in many animal models of brain injury, including hypoxic-ischemic (HI) encephlopathy, trauma and excitotoxicity, but limited data are available for those during the neonatal periods. Here we investigated whether dizocilpine can protect the developing rat brain from HI injury via anti-apoptosis. Methods: In an in vitro model, embryonic cortical neuronal cell culture of Sprague-Dawley (SD) rats at 18-day gestation was done. The cultured cells were divided into three groups: normoxia (N), hypoxia (H), and hypoxia treated with dizocilpine (HD). The N group was prepared in 5% $CO_2$ incubators and the other groups were placed in 1% $O_2$ incubators (94% N2, 5% $CO_2$) for 16 hours. In an in vivo model, left carotid artery ligation was done in 7-day-old SD rat pups. The animals were divided into six groups; hypoxia (N), hypoxia (H), hypoxia with sham-operation (HS), hypoxia with operation (HO), HO treated with vehicle (HV), and HO treated with dizocilpine (HD). Hypoxia was made by exposure to a 2 hour period of hypoxic incubator (92% N2, 8% $O_2$). Results: In the in vitvo and in vivo models, the expressions of Bcl-2 in the hypoxia groups were reduced compared to the normoxia group. whereas those in the dizocilpine-treated group were increased compared to the hypoxia group. However. the expressions of Bax and caspase-3 and the ratio of Bax/Bcl-2 were revealed reversely. Conclusion: Dizocilpine has neuroprotective property over perinatal HI brain injury via anti-apoptosis.