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Iron is an essential element for neuronal as well as cellular functions. However, Iron overload has been known to cause neuronal toxicity through mitochondrial fission, dysregulation of Ca 2+ , endoplasmic reticulum (ER) stress, and reactive oxygen species (ROS) production. Nevertheless, the precise mechanisms of iron-induced oxidative stress and mitochondria- and ER-related iron toxicity in neuronal cells are not fully understood. In this study, we demonstrated that iron overload induces ROS production earlier in the ER than in the mitochondria, and peroxiredoxin 5 (Prx5), which is a kind of antioxidant induced by iron overload, prevents iron overload-induced mitochondrial fragmentation mediated by contact with ER and translocation of Drp1, by inhibiting ROS production and calcium/calcineurin pathway in HT-22 mouse hippocampal neuronal cells. Moreover, Prx5 also prevented iron overload-induced ER-stress and cleavage of caspase-3, which consequently attenuated neuronal cell death. Therefore, we suggested that iron overload induces oxidative stress in the ER earlier than in the mitochondria, thereby increasing ER stress and calcium levels, and consequently causing mitochondrial fragmentation and neuronal cell death. So we thought that this study is essential for understanding iron toxicity in neurons, and Prx5 may serve as a new therapeutic target to prevent iron overload-induced diseases and neurodegenerative disorders., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Reactive oxygen species generated under oxidative stress are involved in neuronal diseases, including ischemia. Glutathione S-transferase pi (GSTpi) is a member of the GST family and is known to play important roles in cell survival. We investigated the effect of GSTpi against oxidative stress-induced hippocampal HT-22 cell death, and its effects in an animal model of ischemic injury, using a cell-permeable PEP-1-GSTpi protein. PEP-1-GSTpi was transduced into HT-22 cells and significantly protected against H2O2-treated cell death by reducing the intracellular toxicity and regulating the signal pathways, including MAPK, Akt, Bax, and Bcl-2. PEP-1-GSTpi transduced into the hippocampus in animal brains, and markedly protected against neuronal cell death in an ischemic injury animal model. These results indicate that PEP-1-GSTpi acts as a regulator or an antioxidant to protect against oxidative stressinduced cell death. Our study suggests that PEP-1-GSTpi may have potential as a therapeutic agent for the treatment of ischemia and a variety of oxidative stress-related neuronal diseases. [BMB Reports 2016; 49(7): 382-387].

Oxidative stress is considered a major factor in various neuronal diseases including ischemia-reperfusion injury. Proviral Integration Moloney 2 (PIM2) proteins, one of the families of PIM kinases, play crucial roles in cell survival. However, the functions of PIM2 protein against ischemia are not understood. Therefore, the protective effects of PIM2 against oxidative stress-induced hippocampal HT22 cell death and brain ischemic injury were evaluated using Tat-PIM2, a cell permeable fusion protein. Tat-PIM2 protein transduced into hippocampal HT22 cells. Low doses of transduced Tat-PIM2 protein protected against oxidative stress-induced cell death including DNA damage and markedly inhibited the activation of mitogen activated protein kinase (MAPKs), NF-κB and the expression levels of Bax protein. Furthermore, Tat-PIM2 protein transduced into the CA1 region of the hippocampus and significantly prevented neuronal cell death in an ischemic insult animal model. These results indicated that low doses of Tat-PIM2 protein protects against oxidative stress-induced neuronal cell death, suggesting low doses of Tat-PIM2 protein provides a potential therapeutic agent against oxidative stress-induced neuronal diseases including ischemia., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To comprehend the role of pyridoxal 5'-phosphate (PLP) in epilepsy or seizure, we investigated whether the expressions of two PLP synthetic enzymes (pyridoxal kinase, PLK; pyridoxine-5'-phosphate oxidase, PNPO) are altered in the hippocampus and whether changes in paired-pulse responses in the hippocampus are associated with altered PLP synthetic enzyme expressions following status epilepticus (SE). PLK and PNPO immunoreactivities were significantly increased in the rat hippocampus accompanied by reductions in paired-pulse inhibition at 1 day and 1 week after SE. Four weeks after SE, PLK and PNPO immunoreactivities in dentate granule cells were similar to those in control animals, while their immunoreactivities were markedly reduced in Cornu Ammonis 1 (CA1) pyramidal cells due to neuronal loss. Linear regression analysis identified a direct proportional relationship between PLK/PNPO immunoreactivity and normalized population spike amplitude ratio in the dentate gyrus and the CA1 region as excluded the data obtained from 4 weeks after SE. These findings indicate that the upregulation of PLK and PNPO immunoreactivities in principal neurons may not be involved in gamma-aminobutyric acid (GABA)ergic inhibition, but rather in enhanced excitability during epileptogenic periods.

Recently we reported that astroglial loss and subsequent gliogenesis in the dentate gyrus play a role in epileptogenesis following pilocarpine-induced status epilepticus (SE). In the present study we investigated whether astroglial damages in the hippocampo-entorhinal complex following SE are relevant to pathological or electrophysiological properties of temporal lobe epilepsy. Astroglial loss/damage was observed in the entorhinal cortex and the CA1 region at 4 weeks and 8 weeks after SE, respectively. These astroglial responses in the hippocampo-entorhinal cortex were accompanied by hyperexcitability of the CA1 region (impairment of paired-pulse inhibition and increase in excitability ratio). Unlike the dentate gyrus and the entorhinal cortex, CA1 astroglial damage was protected by conventional anti-epileptic drugs. alpha-Aminoadipic acid (a specific astroglial toxin) infusion into the entorhinal cortex induced astroglial damage and changed the electrophysiological properties in the CA1 region. Astroglial regeneration in the dentate gyrus and the stratum oriens of the CA1 region was found to originate from gliogenesis, while that in the entorhinal cortex and stratum radiatum of the CA1 region originated from in situ proliferation. These findings suggest that regional specific astroglial death/regeneration patterns may play an important role in the pathogenesis of temporal lobe epilepsy., (Copyright 2008 Wiley-Liss, Inc.)

Since the correlation between the serotonin (5-hydroxytryptamine, 5-HT) system and seizure activity remains to be clarified, we investigated the 5-HT system in the hippocampus of seizure-resistant (SR) and seizure-sensitive (SS) gerbils. There was no difference of the 5-HT system in the hippocampi of young animals (predisposed and juvenile gerbils) in both SR and SS gerbils. 5-HT immunoreactivity in the dorsal raphe nucleus and the median raphe nucleus was also similarly detected in both animal groups. As compared to SR adult gerbils, only 5-HT1A receptor immunoreactivity was selectively reduced in CA1 interneurons within SS adult gerbils. (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (a 5-HT1A receptor agonist, 1 and 2 mg/kg) markedly reduced paired-pulse inhibition in the CA1 region of SS adult gerbils only. These findings suggest that the selective reduction in 5-HT1A receptor expression on CA1 interneurons of SS adult gerbil may not be developmental defects, but be an acquired compensatory change induced by repeated seizure activity.

The epileptic hippocampus shows differential paired-pulse responses between the dentate gyrus and the CA1 region. However, little data are available to explain this phenomenon. In the present study, we identified the relationship between regional differences of paired-pulse response and voltage gated Cl(-) channel 2 (CLC-2)/vesicular GABA transport (VGAT) expression in a pilocarpine-induced rat model. During epileptogenic periods, paired-pulse inhibitions in the dentate gyrus and the CA1 region were markedly reduced. After recurrent seizure onset, paired-pulse inhibition in the dentate gyrus was markedly enhanced, while that in the CA1 region more reduced. Unlike VGAT, CLC-2 immunoreactivity was markedly reduced in the hippocampus during epileptogenic periods and was re-enhanced only in the dentate gyrus after recurrent seizure onset. Linear regression analysis showed an inverse proportional relationship between alterations in CLC-2 immunoreactivity and changes in normalized population spike amplitude ratio within the CA1 region and the dentate gyrus. Therefore, our findings suggest that the regionally specific alterations in CLC-2 immunoreactivity after SE may determine the properties of paired-pulse responses in the hippocampus of the pilocarpine-induced rat epilepsy model.

In the present study, we investigated expressions of vesicular glutamate transporter (VGLUT) and of the plasma membrane glutamate transporters [glutamate transporter 1 (GLT-1), glutamate/aspartate transporter (GLAST) and excitatory amino acid carrier 1 (EAAC-1)] in the gerbil hippocampus following transient ischaemia. The expressional levels and distribution patterns of VGLUT immunoreactivities were unaltered until 3 days after ischaemic-insults. However, VGLUT-2 immunoreactivity in the CA1 region was reduced at 4 days after ischaemia due to delayed neuronal death. In addition, both GLT-1 and GLAST immunoreactivities in the CA1 region were enhanced at 30 min - 12 h after ischaemia-reperfusion and their expression began to reduce at 24 h after ischaemia-reperfusion. In contrast, EAAC-1 immunoreactivity was transiently reduced in the CA1 region at 30 min after ischaemia, re-enhanced at 3-12 h after ischaemia, and re-reduced at 24 h after ischaemia. These findings suggest that malfunctions of plasma membrane glutamate transporters, not of VGLUT, may play an important role in the elevation of extracellular glutamate concentration following ischaemic insults.

K+ -Cl- cotransporter (KCC) plays an important role in maintaining neuronal activity. However, the effect of seizure activity or pharmacological manipulation of GABAergic transmission on KCC expression remains to be clarified. Therefore, the present study was performed to investigate whether seizure activity or GABA receptor agonist treatment changes KCC expression in the gerbil hippocampus. Furthermore, the effect of blockade of KCC on inhibitory transmission in the dentate gyrus was identified following applications of GABA receptor agonists. The distribution of KCC immunoreactivity in the hippocampus was similarly detected between seizure-resistant (SR) and seizure-sensitive (SS) gerbils. Baclofen (a GABAB receptor agonist) treatment markedly increased KCC expression in the gerbil hippocampus. Baclofen treatment significantly reduced paired-pulse inhibition in the dentate gyrus. Furosemide (a KCC inhibitor) treatment amplified the effect of baclofen on paired-pulse responses. In contrast, muscimol (a GABAA receptor agonist) treatment reduced KCC expression. Enhanced paired-pulse inhibition by muscimol treatment was not affected by furosemide treatment. These findings suggest that seizure activity in the gerbil may not affect KCC expression in the hippocampus. In addition, altered KCC immunoreactivity induced by baclofen or muscimol may play an important role in maintaining or regulating inhibitory transmission during GABA receptor activation.

Calcium-binding proteins (CBPs), such as parvalbumin and calbindin D-28k, are useful markers of specific neuronal types in the CNS. In recent studies, expression of CBPs may be indicative of a deactivated neuronal state, particularly epilepsy. However, it is controversial whether altered expression of CBPs in the hippocampus practically indicate neuronal activity. Therefore, the present study was performed to investigate the extent of profiles of expression of CBPs in the rat hippocampus affected by several episodes induced by electroconvulsive shock. In the present study, following electroconvulsive shock expression of CBPs were reduced in the hippocampus in a stimulus-dependent manner, and recovered to the control level at 6 h after electroconvulsive shock. However, paired-pulse responses of the dentate gyrus were transiently impaired by electroconvulsive shock, and immediately normalized to baseline value. In addition, effects of electroconvulsive shock on expression of CBPs and paired-pulse responses were prevented by pretreatment of vigabatrin. These findings suggest that reduced expression of CBPs induced by seizure activity may be indicative of hyperactivity of CBP positive neurons, which is a practical consequence of the abnormal discharge, and that they may play an important role in regulating seizure activity.

In the present study, age-related changes of pyridoxal 5'-phosphate (PLP) synthesizing enzymes, pyridoxal kinase (PLK) and pyridoxine 5'-phosphate oxidase (PNPO), their protein contents and activities were examined in the gerbil hippocampus proper. Significant age-dependent changes in PLK and PNPO immunoreactivities were found in the CA1 region, but not in the CA2/3 region. In the postnatal month 1 (PM 1) group, PLK and PNPO immunoreactivities were detected mainly in the stratum pyramidale of the CA1 region. PLK and PNPO immunoreactivities and their protein contents were highest in the PM 6 group, showing that many CA1 pyramidal cells had strong PLK and PNPO immunoreactivities. Thereafter, PLK and PNPO immunoreactivities started to decrease and were very low at PM 24. Alterations in the change patterns in protein contents and total activities of PLK and PNPO corresponded to the immunohistochemical data, but their specific activities were not altered in any experimental group. Based on double immunofluorescence study, PLK and PNPO immunoreactive cells in the strata oriens and radiatum were identified as GABAergic cells. Therefore, decreases of PLK and PNPO in the hippocampal CA1 region of aged brains may be involved in aging processes related with gamma-aminobutyric acid (GABA) function.

In the present study, the change of vesicular glutamate transporter (VGLUT) immunoreactivity on long-term impaired excitability in the hippocampus after recovery from unilateral hypoxic-ischemic insult was investigated in order to extend our understanding of the mechanism of epileptogenesis using unilateral hypoxic epilepsy models. Both the lesioned (submitted to ischemia) and the unlesioned hippocampi exhibited the frequent occurrence of interictal spikes and occasionally the sustained ictal discharges. However, paired-pulse inhibition was significantly reduced in the unlesioned dentate gyrus, not in the lesioned dentate gyrus. VGLUT1 immunoreactivity was significantly elevated in both hippocampi following hypoxic ischemia, although VGLUT2 immunodensity was unaltered. These findings suggest that the enhancement of VGLUT1 immunoreactivity in both hippocampi after unilateral hypoxic ischemia may contribute to the hyperexcitability, which may play an important role in the epileptogenesis (presumably accompanied by altered inhibitory transmission) after neurodegeneration.

In the present study, we investigated the chronological alterations in SOD1 and its copper chaperone (chaperone for superoxide dismutase, CCS) immunoreactivities and their neuroprotective effects against neuronal damage in the gerbil hippocampus after 5 min of transient forebrain ischemia. SOD1 and CCS immunoreactivities were significantly increased in the stratum pyramidale of the CA1 region at 24 and 12 h after ischemic insult, respectively. At 24 h after ischemic insult, the SOD1 and CCS immunoreactivities were colocalized in the CA1 pyramidal cells of the stratum pyramidale. Thereafter, their immunoreactivities were significantly decreased in the CA1 region. To elucidate the effects of CCS or CCS/SOD1, we constructed the expression vectors PEP-1-SOD and PEP-1-CCS. In the CCS-treated group and the CCS/SOD1-treated group, 43.9 and 78.9% pyramidal cells, respectively, compared to the sham-operated group, were stained with cresyl violet 5 or 7 days after ischemic insult. The distribution pattern of active astrocytes and microglia in the PEP-CCS/SOD1-treated group 5 days after ischemic insult was similar to that of the sham-operated group. In addition, the SOD activity in the PEP-CCS- or PEP-CCS/SOD1-treated group was maintained by 10 days after ischemic insult. The SOD activity was higher in the PEP-CCS/SOD1-treated group vs the CCS-treated group. These results suggest that the enhanced expression of SOD1 and CCS may be related to compensatory mechanisms against ischemic damage and that cotreatment with CCS and SOD1 has a greater neuroprotective effect than treatment with CCS or SOD1 in isolation.

Although reduced calcium binding protein (CBP) immunoreactivities in the epileptic hippocampus have been well established, it has been controversial that these changes may directly indicate neuronal degeneration. In the present study, therefore, we investigated CBP expressions in the gerbil hippocampus following treatment with gamma-aminobutyric acid (GABA) receptor antagonists in order to assess whether altered CBP expressions are the result of either abnormal excitation or indicative of neuronal damage/degeneration. Seizure-sensitive (SS) gerbils showed a loss/decline of CBP immunoreactivities in some hippocampal neurons as compared with seizure-resistant (SR) gerbils. In muscimol (GABA(A) receptor agonist) treated SS gerbils, expression levels of CBP were enhanced as compared with saline-treated SS gerbils. Bicuculline (a GABA(A) receptor antagonist) treatment markedly reduced CBP immunoreactivities in hippocampal neurons of the SR gerbil. Baclofen (a GABA(B) receptor agonist) treatment increased CBP immunoreactivities in the hippocampus of SS gerbils, although its effect was lower than that of muscimol treatment. Moreover, phaclofen (GABA(B) receptor antagonist) treated SR gerbil showed reduction in calbindin D-28K immunoreactivity, not parvalbumin immunoreactivity, in the hippocampus. These findings therefore suggest that reduced CBP immunoreactivities may be the consequence of abnormal discharge caused by loss of GABAergic inhibition rather than an indication of the neuronal damage/degeneration., (Copyright 2005 Wiley-Liss, Inc.)

Reactive oxygen species (ROS) are implicated in reperfusion injury after transient focal cerebral ischemia. The antioxidant enzyme Cu,Zn-superoxide dismutase (SOD) is one of the major means by which cells counteract the deleterious effects of ROS after ischemia. Recently, we reported that denatured Tat-SOD fusion protein is transduced into cells and skin tissue. Moreover, PEP-1 peptide, which has 21 amino acid residues, is a known carrier peptide that delivers full-length native proteins in vitro and in vivo. In the present study, we investigated the protective effects of PEP-1-SOD fusion protein after ischemic insult. A human SOD gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-SOD fusion protein. The expressed and purified fusion proteins were efficiently transduced both in vitro and in vivo with a native protein structure. Immunohistochemical analysis revealed that PEP-1-SOD injected intraperitoneally (i.p.) into mice can have access into brain neurons. When i.p.-injected into gerbils, PEP-1-SOD fusion proteins prevented neuronal cell death in the hippocampus caused by transient forebrain ischemia. These results suggest that the biologically active intact forms of PEP-1-SOD provide a more efficient strategy for therapeutic delivery in various human diseases related to this antioxidant enzyme or to ROS, including stroke.

The present study was performed to determine whether the effects induced by GABA(B) receptor-acting drugs would be related with the alteration in GABA(B) receptor expression in the hippocampus using Mongolian gerbil, a genetic epilepsy model. The distribution patterns of both GABA(B) receptor 1A/B and GABA(B)receptor 2 immunoreactivities were similarly detected in the hippocampi of normal and seizure-prone gerbils. Following baclofen (GABA(B) receptor agonist) or phaclofen (GABA(B) receptor antagonist) treatment, GABA(B) receptor immunoreactivities were decreased or increased by dose-dependent manners, respectively. Vigabatrin (GABA transaminase inhibitor) or 3-mercaptopropionic acid (GAD inhibitor) treatment did not affect GABA(B) receptor expressions. These findings suggest that GABA(B) receptor expression in the gerbil hippocampus may be altered by baclofen or phaclofen treatment.

In the present study, we investigated site-specific expressions of GABA(A) and GABA(B) receptor subunits in the seizure-sensitive (SS) and seizure-resistant (SR) gerbil hippocampus to elucidate the function of the gamma-aminobutyric acid (GABA) receptor in seizure activity in this animal. There were no differences of the immunoreactivities of GABA(B) receptor and some GABA(A) receptor subunits (alpha3, alpha4, pan beta and delta) in the hippocampus between SR and SS gerbils. The alpha1 subunit expression was mainly detected in interneurons of stratum radiatum and hilar region of dentate gyrus in the SR gerbil. However, in SS gerbil, interneurons were nearly devoid of alpha1 subunit immunoreactivity and mainly detected in the molecular layer of dentate gyrus. In SR gerbil, alpha2 subunit immunoreactivity was detected in Ammon's horn, particularly in the CA2 region. In SS gerbil, granule cell layer of the dentate gyrus in SS gerbil showed strong alpha2 subunit immunoreactivity. The distribution of alpha5 and gamma2 subunit immunoreactivity in the hippocampus was similarly detected in SR and SS gerbil. However, alpha5 immunodensity of SR gerbil was slightly lower than that of SS gerbil in CA1 region and was slightly strong than that of SS gerbil in subiculum. These differences in distribution of GABA(A) receptor, not GABA(B) receptor, in the SR and SS gerbil hippocampus may indicate that abnormal hyperactive neuronal discharges are occurred in SS gerbil, which presumably result in spontaneous and repetitive seizure activity in this animal.

To identify the effects of vigabatrin (VGB) on the metabolism of pyridoxal 5'-phosphate (PLP) in the seizure prone gerbil hippocampus, we conducted a chronological and comparative analysis of pyridoxal kinase (PLK) and pyridoxine-5'-phosphate oxidase (PNP oxidase) expression. In the VGB treated animals, PNP oxidase immunoreactivity was reduced, although the distribution and immunodensity of PLK were unaltered, as compared with control animals. In a Western blot study, the densities of PNP oxidase immunoreactivities in VGB treated animals were found to have decreased significantly. However, no differences in PLK immunoreactive bands were observed in controls or in VGB treated animals. By enzyme activity assay, and in contrast to PLK, the specific activity of PNP oxidase in the VGB treated gerbils was significantly reduced. In conclusion, the present data presents a piece of in vivo evidence that supports the anti-epileptic effects mediated by pyridoxamine-5'-phosphate (PMP) metabolism, and which may be helpful in the development of an anti-epileptic drug.

In the previous study, we observed chronological alterations of glutamic acid decarboxylase (GAD), which is the enzyme converting glutamate into GABA. GAD isoforms (GAD65 and GAD67) differ substantially in their interactions with cofactor pyridoxal 5'-phosphate, which is catalyzed by pyridoxal kinase (PLK). In the present study, we examined the chronological changes of PLK expression and activity in the hippocampus after 5 min transient forebrain ischemia in gerbils. PLK immunoreactivity in the sham-operated group was detected weakly in the hippocampus. Ischemia-related change of PLK immunoreactivity in the hippocampus was significant in the hippocampal cornu ammonis (CA1)region, not in the hippocampal CA2/3 region and dentate gyrus. PLK immunoreactivity was observed in non-pyramidal GABAergic neurons at 30 min to 3 h after ischemic insult. At 12 h after ischemic insult, PLK immunoreactivity was shown in many CA1 pyramidal cells as well as some non-pyramidal cells. At this time point, PLK immunoreactivity and protein content was highest after ischemia. Thereafter, PLK immunoreactivity and protein content is decreased time-dependently by 4 days after ischemic insult. Four days after ischemia, some astrocytes expressed PLK in the CA1 region. The specific PLK activity was not altered following ischemic insult up to 2 days after ischemic insult. Thereafter, the specific PLK activity decreased time-dependently. However, total activity of PLK was significantly increased 12-24 h after ischemic insult, and thereafter total activity of PLK decreased. Therefore, we suggest that the over-expression of PLK in the CA1 pyramidal cells at 12 h after ischemia may induce increase of GAD in the CA1 pyramidal cells, which plays an important role in delayed neuronal death via the increase of GABA or enhancement of GABA shunt pathway.

We investigated the distribution of somatostatin receptors (SSTs) in the hippocampi of SR (seizure-resistant) and SS (seizure-sensitive) gerbils in order to characterize the alterations in SST expressions induced by seizure activity. SST2A immunodensity in the hippocampus of SS gerbils was lower than that of SR gerbils, though its localization in the hippocampus was similar in both SR and SS gerbils. SST3 immunodensity in the hippocampus of SS gerbils was lower than in SR gerbils. In SR gerbils, strong SST4 immunoreactivity was detected in the dentate gyrus and in the CA3 region, in contrast little immunoreactivity was detected in these regions in SS gerbils. In SR and SS gerbils, the strong SST5 immunoreactivity in the hippocampus was also detected in the stratum oriens of the CA2-3 regions and the septal area of CA1 region. However, SST5 immunodensity in the stratum radiatum in SS gerbils was lower than in SR gerbils. These results are the first comprehensive description of the distribution of SSTs in the normal and epileptic hippocampus of gerbils, and suggest that these alterations in the hippocampus of the SS gerbil may be related with a regulatory mechanism for seizure activity in these seizure prone animals.

Vigabatrin (VGB, gamma-vinyl-gamma-aminobutyric acid (GABA)), an irreversible inhibitor of GABA transaminase, increases regional inhibitory effects by elevating GABA concentration and reducing glutamate synthesis. In the present study, changes in glutamate dehydrogenase (GDH) activity and its immunoreactivity in the seizure prone gerbil hippocampus after treating VGB were investigated to identify the effect of VGB on energy and/or glutamate metabolism via GDH. In the VGB treated group, GDH immunoreactivity and its activity in the hippocampus were significantly decreased, as compared with those of controls. These findings suggest that VGB administration may suppress the development and spread of seizures not only by elevating the level of GABA, but also by affecting the glutamate signaling and energy metabolism in neurons., (Copyright 2003 Elsevier Science Ireland Ltd.)

Gastrodin is one of the natural compound isolated from Gastrodia elata and has known anticonvulsant effects, although the exact pharmacological principles of this natural compound and its effects on other aspects of gamma-aminobutyric acid (GABA) metabolism in vivo have not been explored. Therefore, in the present study, the effects of gastrodin on GABA metabolism in the gerbil hippocampus were examined, in an effort to identify the antiepileptic characteristics of this substance. Gastrodin reduced the seizure score in the treated group, although the immunoreactivities of GABA synthetic enzymes and GABA transporters were unaltered in gastrodin-treated animals. Interestingly, in the gastrodin-treated group, GABA transaminase (GABA-T) immunoreactivity in the hippocampus, particularly in neurons, was significantly decreased. In the gastrodin-treated group, both succinic semialdehyde dehydrogenase (SSADH) and succinic semialdehyde reductase (SSAR) immunoreactivities in the hippocampus was also decreased significantly, which stood in contrast to the nontreated group, in which strong SSADH and SSAR immunoreactivities were detected. From the neuroanatomical viewpoint, these findings suggest that gastrodin may cause the elevation of GABA concentration by inhibiting the GABA shunt., (Copyright 2002 Wiley-Liss, Inc.)

In the present study, the distribution of succinic semialdehyde dehydrogenase (SSADH) and succinic semialdehyde reductase (SSAR) in the hippocampus of the Mongolian gerbil and its association with various sequelae of spontaneous seizure were investigated in order to identify the roles of GABA shunt in the epileptogenesis and the recovery mechanisms in these animals. Both SSADH and SSAR immunoreactivities in the GABAergic neurons were significantly higher in the pre-seizure groups of seizure sensitive (SS) gerbil as compared to those seen in the seizure resistant (SR) gerbils. The distributions of both SSADH and SSAR immunoreactivities in the hippocampus showed significant differences after the on-set of seizure. At 3 h postictal, when compared to the pre-seizure group of SS gerbils, a decline in the immunoreactivities in the perikarya was observed. At 12 h after seizure on-set, the densities of both SSADH and SSAR immunoreactivities were begun to recover to the pre-seizure level of SS gerbils. These results suggest that the GABAergic neurons in the hippocampal complex of the SS gerbil may be highly activated. In addition, the imbalance of GABA shunt expressions in the GABAergic neurons may imply a malfunction of the metabolism of GABAergic neurons in the SS gerbils, and this defect may trigger seizure on-set. Therefore, the initiation of seizure, at least in gerbils, may be the result of a malfunction in GABA shunt in the GABAergic neurons., (Copyright 2003 Elsevier Science Ltd.)

To identify the roles of pyridoxal kinase (PLK) in epileptogenesis and the recovery mechanisms in spontaneous seizure, a chronological and comparative analysis of PLK expression in the gerbil hippocampus was conducted. PLK immunoreactivity in a pre-seizure group of seizure sensitive (SS) gerbils was more strongly detected than that in a seizure resistant (SR) group. The density of PLK immunoreactivity in a 30-min postictal group was significantly lower than that of a pre-seizure group. In a 12 h postictal group, PLK immunodensity recovered to pre-seizure level. The over-expression of PLK in the hippocampus of pre-seizure SS gerbils suggests that PLP play an important role in the modulation of GAD activity and GABA reuptake as mediated by membrane transporter via neurons.

To identify the roles of pyridoxine-5'-phosphate (PNP) oxidase in epileptogenesis and the recovery mechanisms in spontaneous seizure, a chronological and comparative analysis of PNP oxidase expression was conducted. PNP oxidase immunoreactivity in a preseizure group of seizure-sensitive (SS) gerbils was detected more strongly than that in a seizure-resistant (SR) group. The density of PNP oxidase immunoreactivity in a 30 min postictal group was significantly lower than that in a preseizure group. In a 12 hr postictal group, PNP oxidase immunodensity had recovered to a preseizure level. The overexpression of PNP oxidase in the hippocampus of preseizure SS gerbils suggests that PNP or pyridoxal 5'-phosphate plays an important role in the modulation of glutamic acid decarboxylase activity and gamma-aminobutyric acid reuptake as mediated by membrane transporter via neurons. In addition, this change in the PNP oxidase immunoreactivity following seizure may be a compensatory response designed to reduce epileptic activity in this animal., (Copyright 2002 Wiley-Liss, Inc.)

In the present study, the distribution of glutamic acid decarboxylase (GAD) isoforms in the hippocampus of the Mongolian gerbil and its association with different sequelae of spontaneous seizure were investigated to identify the roles of balance of GAD isoforms in the epileptogenesis and the recovery mechanisms in these animals. The GAD67/GAD65 ratio in the hippocampus of pre-seizure seizure sensitive (SS) gerbil was approximately 3.5-fold higher as compared to seizure resistant (SR) gerbil. Following seizure, this ratio shifted to the level of SR gerbils up to 12 h postical. Therefore, the mismatched GAD67/GAD65 ratio (imbalance of GAD isoform expressions) in the hippocampus of SS gerbil implies that GABAergic neurons may be highly activated in order to regulate the increased neuronal excitability. In addition, the alteration in this ratio after seizure may be the compensatory response for reduction of epileptic activity in this animal.