• The Journal of Internal Korean Medicine
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• v.25 no.3
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• pp.461-472
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• 2004

Objectives : For the screening of neuroprotective effects of medicinal herbs, the complex system of animal models suffer some disadvantages in controlling critical parameters such as blood pressure and body temperature. Additionally, application of drugs to the appropriate brain area sometimes is difficult, due to poor permeability though the blood brain barrier, and so potential protective effects might be masked. Methods : Organotypic hippocampal slice culture (OHSC) method has the advantages of being relatively easy to prepare and of maintaining the general structure, including tissue integrity and the connections between cells. Drugs can easily be applied and neuronal damage can easily be quantified by using tissues and culture media. This study demonstrates neuroprotective effects of Puerariae radix (葛根, PR), Salviae miltiorrhizae radix (丹蔘, SR), Rhei rhizoma (大黃, RR), and Bupleuri radix (柴胡, BR). These were screenedand compared to MK-801, antagonist of NMDA receptors, by using OHSC of 1 week-old Sprague-Dawley rats. Oxygen/glucose deprivation (OGD) were conducted in an anaerobic chamber $(85%\;N_2,\;10%\;CO_2\;and\;5%\;H_2)$ in a deoxygenated glucose-free medium for 60 minutes. Water extracts of each herbs were treated to culture media with $5\;{\mu}g/ml$ for 48 hours. Results : Neuronal cell death in the cultures was monitored by densitometric measurements of the cellular uptake of propidium iodide (PI). PI fluorescence images were obtained at 48 hours after the OGD and medicinal herb treatment. Also TUNEL-positive cells in the CAI and DG regions and LDH concentrations in culture media were measured at 48 hours after the OGD. According to measured data, MK-801, PR, SR and BR demonstrated significant neuroprotective effect against excessive neuronal cell death and apoptosis induced by the OGD insult. Especially, PR revealed similar neuroprotective effect to MK-801 and RR demonstrated weak neuroprotective effect. Conclusions : These results suggest that OHSC can be a suitable method for screening of neuroprotective effects of medicinal herbs. (This work was supported by the research program of Dongguk University and Grant 01-PJ9-PG1-01CO03-0003 from Ministry of Health & Welfare.)

• Toxicological Research
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• v.14 no.4
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• pp.483-488
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• 1998

During cerebral ischemia two important factors such as hypoxia and reduction of glucose can act as modulating stressor affecting the release of amine neurotransmitters including 5-hydroxytryptamine (5-HT). This study was performed to investigate the effect of glucose deprivation on the oxygen deprivation-induced changes of [3H]-5-HT release in the rat hippocampal slices. Experimental groups were divided into 4 groups for this study: normoxic/normoglycemic group, oxygen-deprived group, glucose-deprived group, and oxygen/glucose-deprived group. The hippocampus of rat brain was sliced by 400 $\mu\textrm{m}$ thickness with manual chopper. After 30 minutes preincubation in the normal buffer, the slices were incubated for 20 min in buffer containing [3H]-5-HT (0.1 M, 74 $\mu\textrm$Ci) for uptake. To measure the release of [3H]-5-HT into the buffer, the incubation medium was drained of and refilled with fresh buffer every ten minutes through a sequence of 14 tubes. Oxygen deprivation by gassing with 95% $N_2$/5% $CO_2$ and/or glucose deprivation was done in the 6th and 7th tube. The radioactivities in each buffer and the tissue were counted using scintillation counter. The results were expressed as fractional release. When slices were exposed to oxygen-deprived media for 20 min, the diminution followed by the rebound release of [3H]-5-HT was observed during the post-oxygen deprived period. However, glucose deprivation or oxygen/glucose deprivation markedly increased the release of [3H]-5-HT. which was opposite to the pattern observed in oxygen-deprived group. These results suggested that oxygen deprivation itself inhibits [3H]-5-HT release in rat hippocampal slices during oxygen-deprived period, but additional glucose deprivation convert the inhibitory response to increase of [3H]-5-HT release.

• Clinical and Experimental Pediatrics
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• v.58 no.4
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• pp.142-147
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• 2015

Purpose: The aim of this study was to investigate the potential effects of mild hypoxia in the mature and immature brain. Methods: We prepared organotypic slice cultures of the hippocampus and used hippocampal tissue cultures at 7 and 14 days in vitro (DIV) to represent the immature and mature brain, respectively. Tissue cultures were exposed to 10% oxygen for 60 minutes. Twenty-four hours after this hypoxic insult, propidium iodide fluorescence images were obtained, and the damaged areas in the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG) were measured using image analysis. Results: In the 7-DIV group compared to control tissue, hypoxia-exposed tissue showed decreased damage in two regions (CA1: $5.59%{\pm}2.99%$ vs. $4.80%{\pm}1.37%$, P=0.900; DG: $33.88%{\pm}12.53%$ vs. $15.98%{\pm}2.37%$, P=0.166), but this decrease was not statistically significant. In the 14-DIV group, hypoxia-exposed tissue showed decreased damage compared to control tissues; this decrease was not significant in the CA3 ($24.51%{\pm}6.05%$ vs. $18.31%{\pm}3.28%$, P=0.373) or DG ($15.72%{\pm}3.47%$ vs. $9.91%{\pm}2.11%$, P=0.134), but was significant in the CA1 ($50.91%{\pm}5.90%$ vs. $32.30%{\pm}3.34%$, P=0.004). Conclusion: Although only CA1 tissues cultured for 14 DIV showed significantly less damage after exposure to hypoxia, the other tissues examined in this study showed a tendency towards less damage after hypoxic exposure. Therefore, mild hypoxia might play a protective role in the brain.

• Clinical and Experimental Pediatrics
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• v.49 no.5
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• pp.558-564
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• 2006

Purpose : Transcranial electromagnetic stimulation(TMS) is a noninvasive method which stimulates the central nervous system through pulsed magnetic fields without direct effect on the neurons. Although the neurobiologic mechanisms of magnetic stimulation are unknown, the effects on the brain are variable according to the diverse stimulation protocols. This study aims to observe the effect of the magnetic stimulation with two different stimulation methods on the cultured hippocampal slices. Methods : We obtained brains from 8-days-old Spague-Dawley rats and dissected the hippocampal tissue under the microscope. Then we chopped the tissue into 450 µm thickness slices and cultured the hippocampal tissue by Stoppini's method. We divided the inserts, which contained five healthy cultured hippocampal slices respectively, into magnetic stimulation groups and a control group. To compare the different effects according to the frequency of magnetic stimulation, stimulation was done every three days from five days in vitro at 0.67 Hz in the low stimulation group and at 50 Hz in the high stimulation group. After N-methyl-D-aspartate exposure to the hippocampal slices at 14 days in vitro, magnetic stimulation was done every three days in one and was not done in another group. To evaluate the neuronal activity after magnetic stimulation, the $NeuN/{\beta}$-actin ratio was calculated after western blotting in each group. Results : The expression of NeuN in the magnetic stimulation group was stronger than that of the control group, especially in the high frequency stimulation group. After N-methyl-D-aspartate exposure to hippocampal slices, the expression of NeuN in the magnetic stimulation group was similar to that of the control group, whereas the expression in the magnetic non-stimulation group was lower than that of the control group. Conclusion : We suggest that magnetic stimulation increases the neuronal activity in cultured hippocamal slices, in proportion to the stimulating frequency, and has a neuroprotective effect on neuronal damage.

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.2
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• pp.87-91
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• 2002

The aim of this study was to investigate the role of $Ca^{2+}-channel$ blockers in norepinephrine (NE) release from rat hippocampus. Slices and synaptosomes were incubated with $[^3H]-NE$ and the releases of the labelled products were evoked by 25 mM KCl stimulation. Nifedipine, diltiazem, nicardipine, flunarizine and pimozide did not affect the evoked and basal release of NE in the slice. But, diltiazem, nicardipine and flunarizine decreased the evoked NE release with a dose-related manner without any change of the basal release from synaptosomes. Also, a large dose of pimozide produced modest decrement of NE release. ${\omega}-conotoxin$ (CTx) GVIA decreased the evoked NE release in a dose-dependent manner without changing the basal release. And ${\omega}-CTxMVIIC$ decreased the evoked NE release in the synaoptosomes without any effect in the slice, but the effect of decrement was far less than that of ${\omega}-CTxGVIA.$ In interaction experiments with ${\omega}-CTxGVIA,\;{\omega}-CTxMVIIC$ slightly potentiated the effect of ${\omega}-CTxGVIA$ on NE release in the slice and synaptosomal preparations. These results suggest that the NE release in the rat hippocampus is mediated mainly by N-type $Ca^{2+}-channels,$ and that other types such as L-, T- and/or P/Q-type $Ca^{2+}-channels$ could also be participate in this process.

• Journal of the korean academy of Pediatric Dentistry
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• v.27 no.1
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• pp.7-14
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• 2000

Inhibitory cells are critically involved in shaping normal hippocampal function and are thought to be important elements in the development of hippocampal pathologies. The present study was carried out in hippocampal CA1 area in vivo to compare with hippocampal slice studies. Intracellular and extracellular recordings with or without bicuculline electrodes were obtained in the intact brain of anesthetized rats, and cells were intracellularty labelled with neurobiotin. Electrical stimulation of fimbria-fornix resulted in an initial short-latency population spike. In the presence of $10{\mu}M$ bicuculline, orthodromic stimulation resulted in bursts of population spikes. The amplitude of population spikes in the CA1 region increased with stimulus intensity, as did the number of population spikes when the field recording electrode contained $10{\mu}M$ bicuculline. We measured the level of excitability in the CA1 area, using a paired-pulse stimulus paradigm to evoke population spikes. Population spikes showed strong paired-pulse inhibition at short interstimulus intervals. Burst afterdischarges up to 400 ms were observed after paired-pulse stimulus. These result suggest that hippocampal CA1 inhibitory interneurons can affect the excitability of pyramidal neurons that can not be appreciated in conventional in vitro preparation.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.6
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• pp.457-466
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• 2001

Glutamate is the most common excitatory amino acid in the brain. Responsiveness of dendrites to the glutamate greatly varies depending on the application sites. Especially, a point of the maximal response to the glutamate of the dendrite is called as 'hot spot'. In our experiment, the responsiveness of the hot spot to the glutamate was investigated in the CA1 pyramidal neuron of the rat hippocampal slice. CNQX, the antagonist of AMPA receptor, blocked 95% of membrane current to the glutamate focal application $(I_{gl}).$ Train ejection of glutamate on one point of the dendrite increased or decreased the amplitude of $I_{gl}$ with the pattern of train, and the changes were maintained at least for 30 min. In some cases, glutamate train ejection also induced calcium dependent action potentials. To evoke long-term change of synaptic plasticity, we adopted ${\theta}-burst$ in the glutamate train ejection. The ${\theta}-burst$ decreased the amplitude of glutamate response by 60%. However, after ${\theta}-burst$ glutamate train ejection, the calcium dependent action potential appeared. These results indicated that the focal application of glutamate on the neuronal dendrite induced response similar to the synaptic transmission and the trains of glutamate ejection modulated the change of AMPA receptor.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.6
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• pp.657-664
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• 1997

The effects of adenosine, adenosine A1 receptor antagonist (DPCPX), or NMDA receptor antagonist (APV) on the spontaneous release of $[^3H]-5-hydroxytryptamine$ ($[^3H]-5-HT$) during normoxic/normoglycemic or hypoxic/hypoglycemic period were studied in the rat hippocampal slices. The hippocampus was obtained from the rat brain and sliced $400\;{\mu}m$ thickness with the tissue slicer. After 30 min's preincubation in the normal buffer, the slices were incubated for 30 min in a buffer containing $[^3H]-5-HT$ ($0.1\;{\mu}M,\;74{\mu}Ci/8\;ml$) for uptake, and washed. To measure the release of $[^3H]-5-HT$ into the buffer, the incubation medium was drained off and refilled every ten minutes through sequence of 14 tubes. Induction of glucose/oxygen deprivation (GOD; medium depleting glucose and gassed with 95% $N_2/5%\;CO_2$) was done in 6th and 7th tube. The radioactivities in each buffer and the tissue were counted using liquid scintillation counter and the results were expressed as a percentage of the total radioactivities. When slices were exposed to GOD for 20 mins, the spontaneous release of $[^3H]-5-HT$ was markedly increased and this increase of $[^3H]-5-HT$ release was blocked by adenosine ($10\;{\mu}M$) or DL-2-amino-5-phosphonovaleric acid (APV; $30\;{\mu}M$). Adenosine $A_1$ receptor specific antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) exacerbate GOD-induced increase of spontaneous release of $[^3H]-5-HT$. These results suggest that Adenosine may play a role in the GOD-induced spontaneous release of $[^3H]-5-HT$ through adenosine $A_1$ receptor activity.

• Clinical and Experimental Pediatrics
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• v.45 no.12
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• pp.1551-1558
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• 2002

Purpose : Loss of hippocampal interneurons in dentate gyrus has been reported in patients with severe temporal lobe epilepsy and in animals treated with kainic acid(KA). Interneurons contain $Ca^{2+}$- binding protein parvalbumin(PV). The effects of kainic acid on parvalbumin-immunoreactive (PV-IR) interneurons in dentate gyrus were investigated in organotypic hippocampal slice cultures. Methods : Cultured hippocampal slices from postnatal day nine C57/BL6 mice were exposed to $10{\mu}M$ KA, and were observed at 0, 8, 24, 48, 72 hours after a one hour KA exposure. Neuronal injury was determined by morphologic changes of PV-IR interneuron in dentate gyrus. Results : Transient(1 hour) exposure of hippocampal explant cultures to KA produced marked varicosities in dendrites of PV-IR interneuron in dentate gyrus and the shaft of interbeaded dendrite is often much thinner than those in control. The presence of varicosities in dendrites was reversible with KA washout. The dendrites of KA treated explants were no longer beaded at 8, 24, 48 and 72 hours after KA exposure. The number of cells in PV-IR interneurons in dentate gyrus was decreased at 0, 8 hours after exposure. But there was no significant difference in 24, 48 and 72 hours recovery group compared with control group. Conclusion : The results suggested that loss of PV-IR interneurons in dentate gyrus is transient, and is not accompanied by PV-IR interneuronal cell death.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.5
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• pp.467-475
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• 1997

Although one of the major physiological functions of taurine(2-aminoethanesulfonic acid) is the inhibitory action on the central nervous system(CNS), the mechanism of taurine in controlling the neuronal excitation in the CNS has been in controversy. Electrically evoked pEPSP and spontaneous activity induced by the perfusion of low $Mg^{++}-ACSF$ were recorded in the CA1 pyramidal cell layer of the hippocampal slice. To test the inhibitory effect of taurine on spontaneous responses, taurine was treated for 2 min at various concentrations(1 mM-10 mM). Taurine reduced the spontaneous activity by 22.2% at 1 mM, and 100% at 2 mM in low $Mg^{++}-ACSF$. Evoked response was induced by electrical stimulation of Schaffer collateral-commissural fibers. Taurine reduced the evoked response by 11.68% at 3 mM, and 24.25% at 5 mM. Even 20 mM of taurine reduced the evoked response only by 24 % after 5 min treatment. That is, the inhibitory efficacy was much higher in spontaneous activity than in evoked response. The $GABA_A$ receptor antagonist, 100 uM bicuculline, blocked the inhibitory action of taurine, while $GABA_B$ receptor antagonist, 700 uM phaclofen, did not. Taurine blocked the spontaneous activity in the presence of CNQX, and did not block the electrically evoked responce in the presence of APV. The results suggest that taurine causes hyperpolarization in the cell by binding to $GABA_A$ receptor and preferentially attenuates NMDA receptor-mediated hyperexcitation, leaving synaptic transmission unmodified.