Spatiotemporal expression of RCAN1 and its isoform RCAN1-4 in the mouse hippocampus after pilocarpine-induced status epilepticus

Regulator of calcineurin 1 (RCAN1) can be induced by an intracellular calcium increase and oxidative stress, which are characteristic features of temporal lobe epilepsy. Thus, we investigated the spatiotemporal expression and cellular localization of RCAN1 protein and mRNA in the mouse hippocampus after pilocarpine-induced status epilepticus (SE). Male C57BL/6 mice were given pilocarpine hydrochloride (280 mg/kg, i.p.) and allowed to develop 2 h of SE. Then the animals were given diazepam (10 mg/kg, i.p.) to stop the seizures and sacrificed at 1, 3, 7, 14, or 28 day after SE. Cresyl violet staining showed that pilocarpine-induced SE resulted in cell death in the CA1 and CA3 subfields of the hippocampus from 3 day after SE. RCAN1 immunoreactivity showed that RCAN1 was mainly expressed in neurons in the shammanipulated hippocampi. At 1 day after SE, RCAN1 expression became detected in hippocampal neuropils. However, RCAN1 signals were markedly enhanced in cells with stellate morphology at 3 and 7 day after SE, which were confirmed to be reactive astrocytes, but not microglia by double immunofluorescence. In addition, real-time reverse transcriptase–polymerase chain reaction showed a significant upregulation of RCAN1 isoform 4 (RCAN1-4) mRNA in the SE-induced hippocampi. Finally, in situ hybridization with immunohistochemistry revealed astrocytic expression of RCAN1-4 after SE. These results demonstrate astrocytic upregulation of RCAN1 and RCAN1-4 in the mouse hippocampus in the acute and subacute phases of epileptogenesis, providing foundational information for the potential role of RCAN1 in reactive astrocytes during epileptogenesis.

Increased expression of vascular endothelial growth factor-C and vascular endothelial growth factor receptor-3 after pilocarpine-induced status epilepticus in mice

Vascular endothelial growth factor (VEGF)-C and its receptor, vascular endothelial growth factor receptor (VEGFR)-3, are responsible for lymphangiogenesis in both embryos and adults. In epilepsy, the expression of VEGF-C and VEGFR-3 was significantly upregulated in the human brains affected with temporal lobe epilepsy. Moreover, pharmacologic inhibition of VEGF receptors after acute seizures could suppress the generation of spontaneous recurrent seizures, suggesting a critical role of VEGF-related signaling in epilepsy. Therefore, in the present study, the spatiotemporal expression of VEGF-C and VEGFR-3 against pilocarpine-induced status epilepticus (SE) was investigated in C57BL/6N mice using immunohistochemistry. At 1 day after SE, hippocampal astrocytes and microglia were activated. Pyramidal neuronal death was observed at 4 days after SE. In the subpyramidal zone, VEGF-C expression gradually increased and peaked at 7 days after SE, while VEGFR-3 was significantly upregulated at 4 days after SE and began to decrease at 7 days after SE. Most VEGF-C/VEGFR-3-expressing cells were pyramidal neurons, but VEGF-C was also observed in some astrocytes in sham-manipulated animals. However, at 4 days and 7 days after SE, both VEGFR-3 and VEGF-C immunoreactivities were observed mainly in astrocytes and in some microglia of the stratum radiatum and lacunosum-moleculare of the hippocampus, respectively. These data indicate that VEGF-C and VEGFR-3 can be upregulated in hippocampal astrocytes and microglia after pilocarpine-induced SE, providing basic information about VEGF-C and VEGFR-3 expression patterns following acute seizures.

Black Rice (Oryza sativa L., Poaceae) Extract Reduces Hippocampal Neuronal Cell Death Induced by Transient Global Cerebral Ischemia in Mice

Rice is the most commonly consumed grain in the world. Black rice has been suggested to contain various bioactive compounds including anthocyanin antioxidants. There is currently little information about the nutritional benefits of black rice on brain pathology. Here, we investigated the effects of black rice (Oryza sativa L., Poaceae) extract (BRE) on the hippocampal neuronal damage induced by ischemic insult. BRE (300 mg/kg) was orally administered to adult male C57BL/6 mice once a day for 21 days. Bilateral common carotid artery occlusion (BCCAO) was performed for 23 min on the 8th day of BRE or vehicle administration. Histological analyses conducted on the 22nd day of BRE or vehicle administration revealed that administering BRE profoundly attenuated neuronal cell death, inhibited reactive astrogliosis, and prevented loss of glutathione peroxidase expression in the hippocampus when compared to vehicle treatment. In addition, BRE considerably ameliorated BCCAO-induced memory impairment on the Morris water maze test from the 15th day to the 22nd day of BRE or vehicle administration. These results indicate that chronic administration of BRE is potentially beneficial in cerebral ischemia.

Neuroprotective effect of lithium after pilocarpine-induced status epilepticus in mice

Status epilepticus is the most common serious neurological condition triggered by abnormal electrical activity, leading to severe and widespread cell loss in the brain. Lithium has been one of the main drugs used for the treatment of bipolar disorder for decades, and its anticonvulsant and neuroprotective properties have been described in several neurological disease models. However, the therapeutic mechanisms underlying lithium's actions remain poorly understood. The muscarinic receptor agonist pilocarpine is used to induce status epilepticus, which is followed by hippocampal damage. The present study was designed to investigate the effects of lithium post-treatment on seizure susceptibility and hippocampal neuropathological changes following pilocarpine-induced status epilepticus. Status epilepticus was induced by administration of pilocarpine hydrochloride (320 mg/kg, i.p.) in C57BL/6 mice at 8 weeks of age. Lithium (80 mg/kg, i.p.) was administered 15 minutes after the pilocarpine injection. After the lithium injection, status epilepticus onset time and mortality were recorded. Lithium significantly delayed the onset time of status epilepticus and reduced mortality compared to the vehicle-treated group. Moreover, lithium effectively blocked pilocarpine-induced neuronal death in the hippocampus as estimated by cresyl violet and Fluoro-Jade B staining. However, lithium did not reduce glial activation following pilocarpine-induced status epilepticus. These results suggest that lithium has a neuroprotective effect and would be useful in the treatment of neurological disorders, in particular status epilepticus.

Chronic cerebral hypoperfusion and plasticity of the posterior cerebral artery following permanent bilateral common carotid artery occlusion

Vascular dementia (VaD) is a group of heterogeneous diseases with the common feature of cerebral hypoperfusion. To identify key factors contributing to VaD pathophysiology, we performed a detailed comparison of Wistar and Sprague–Dawley (SD) rats subjected to permanent bilateral common carotid artery occlusion (BCCAo). Eight-week old male Wistar and SD rats underwent BCCAo, followed by a reference memory test using a five-radial arm maze with tactile cues. Continuous monitoring of cerebral blood flow (CBF) was performed with a laser Doppler perfusion imaging (LDPI) system. A separate cohort of animals was sacrificed for evaluation of the brain vasculature and white matter damage after BCCAo. We found reference memory impairment in Wistar rats, but not in SD rats. Moreover, our LDPI system revealed that Wistar rats had significant hypoperfusion in the brain region supplied by the posterior cerebral artery (PCA). Furthermore, Wistar rats showed more profound CBF reduction in the forebrain region than did SD rats. Post-mortem analysis of brain vasculature demonstrated greater PCA plasticity at all time points after BCCAo in Wistar rats. Finally, we confirmed white matter rarefaction that was only observed in Wistar rats. Our studies show a comprehensive and dynamic CBF status after BCCAo in Wistar rats in addition to severe PCA dolichoectasia, which correlated well with white matter lesion and memory decline.

The neuroprotective mechanism of ampicillin in a mouse model of transient forebrain ischemia

Ampicillin, a beta-lactam antibiotic, dose-dependently protects neurons against ischemic brain injury. The present study was performed to investigate the neuroprotective mechanism of ampicillin in a mouse model of transient global forebrain ischemia. Male C57BL/6 mice were anesthetized with halothane and subjected to bilateral common carotid artery occlusion for 40 min. Before transient forebrain ischemia, ampicillin (200 mg/kg, intraperitoneally [i.p.]) or penicillin G (6,000 U/kg or 20,000 U/kg, i.p.) was administered daily for 5 days. The pretreatment with ampicillin but not with penicillin G signifi cantly attenuated neuronal damage in the hippocampal CA1 subfield. Mechanistically, the increased activity of matrix metalloproteinases (MMPs) following forebrain ischemia was also attenuated by ampicillin treatment. In addition, the ampicillin treatment reversed increased immunoreactivities to glial fibrillary acidic protein and isolectin B4, markers of astrocytes and microglia, respectively. Furthermore, the ampicillin treatment significantly increased the level of glutamate transporter-1, and dihydrokainic acid (DHK, 10 mg/kg, i.p.), an inhibitor of glutamate transporter-1 (GLT-1), reversed the neuroprotective effect of ampicillin. Taken together, these data indicate that ampicillin provides neuroprotection against ischemia-reperfusion brain injury, possibly through inducing the GLT-1 protein and inhibiting the activity of MMP in the mouse hippocampus.

Neuroprotective effect of caffeic acid phenethyl ester in 3-nitropropionic acid-induced striatal neurotoxicity

Caffeic acid phenethyl ester (CAPE), derived from honeybee hives, is a bioactive compound with strong antioxidant activity. This study was designed to test the neuroprotective effect of CAPE in 3-nitropropionic acid (3NP)-induced striatal neurotoxicity, a chemical model of Huntington's disease (HD). Initially, to test CAPE's antioxidant activity, a 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) antioxidant assay was employed, and CAPE showed a strong direct radical-scavenging eff ect. In addition, CAPE provided protection from 3NP-induced neuronal cell death in cultured striatal neurons. Based on these observations, the in vivo therapeutic potential of CAPE in 3NP-induced HD was tested. For this purpose, male C57BL/6 mice were repeatedly given 3NP to induce HD-like pathogenesis, and 30 mg/kg of CAPE or vehicle (5% dimethyl sulfoxide and 95% peanut oil) was administered daily. CAPE did not cause changes in body weight, but it reduced mortality by 29%. In addition, compared to the vehicle-treated group, robustly reduced striatal damage was observed in the CAPE-treated animals, and the 3NP-induced behavioral defi cits on the rotarod test were signifi cantly rescued after the CAPE treatment. Furthermore, immunohistochemical data showed that immunoreactivity to glial fibrillary acidic protein (GFAP) and CD45, markers for astrocyte and microglia activation, respectively, were strikingly reduced. Combined, these data unequivocally indicate that CAPE has a strong antioxidant eff ect and can be used as a potential therapeutic agent against HD.

Influence of Aspirin on Pilocarpine-Induced Epilepsy in Mice

Aspirin (acetylsalicylic acid) is one of the most widely used therapeutic agents based on its pharmacological actions, including anti-inflammatory, analgesic, anti-pyretic, and anti-thrombotic effects. In this study, we investigated the effects of aspirin on seizure susceptibility and hippocampal neuropathology following pilocarpine-induced status epilepticus (SE). SE was induced by pilocarpine hydrochloride (280 mg/kg, i.p.) administration in C57BL/6 mice (aged 8 weeks). Aspirin was administered daily (15 mg/kg or 150 mg/kg, i.p.) for 10 days starting 3 days before SE, continuing until 6 days after SE. After pilocarpine injection, SE onset time and mortality were recorded. Neuronal cell death was examined using cresyl violet and Fluoro-Jade staining, and glial responses were observed 7 days post SE using immunohistochemistry. In the aspirin-treated group, the onset time of SE was significantly shortened and mortality was markedly increased compared to the control group. However, in this study, aspirin treatment did not affect SE-induced neuronal cell death or astroglial and microglial responses in the hippocampus. In conclusion, these results suggest that the safety of aspirin should be reevaluated in some patients, especially with neurological disorders such as temporal lobe epilepsy.

The Neuroprotective Potential of Cyanidin-3-glucoside Fraction Extracted from Mulberry Following Oxygen-glucose Deprivation

In this study, cyanidin-3-glucoside (C3G) fraction extracted from the mulberry fruit (Morus alba L.) was investigated for its neuroprotective effects against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Cell membrane damage and mitochondrial function were assessed by LDH release and MTT reduction assays, respectively. A time-course study of OGD-induced cell death of primary cortical neurons at 7 days in vitro (DIV) indicated that neuronal death was OGD duration-dependent. It was also demonstrated that OGD for 3.5 h resulted in approximately 50% cell death, as determined by the LDH release assay. Treatments with mulberry C3G fraction prevented membrane damage and preserved the mitochondrial function of the primary cortical neurons exposed to OGD for 3.5 h in a concentration-dependent manner. Glutamate-induced cell death was more pronounced in DIV-9 and DIV-11 cells than that in DIV-7 neurons, and an application of 50microM glutamate was shown to induce approximately 40% cell death in DIV-9 neurons. Interestingly, treatment with mulberry C3G fraction did not provide a protective effect against glutamate-induced cell death in primary cortical neurons. On the other hand, treatment with mulberry C3G fraction maintained the mitochondrial membrane potential (MMP) in primary cortical neurons exposed to OGD as assessed by the intensity of rhodamine-123 fluorescence. These results therefore suggest that the neuroprotective effects of mulberry C3G fraction are mediated by the maintenance of the MMP and mitochondrial function but not by attenuating glutamate-induced excitotoxicity in rat primary cortical neurons.

GS28 Protects Neuronal Cell Death Induced by Hydrogen Peroxide under Glutathione-Depleted Condition

Golgi SNAP receptor complex 1 (GS28) has been implicated in vesicular transport between intra-Golgi networks and between endoplasmic reticulum (ER) and Golgi. Additional role(s) of GS28 within cells have not been well characterized. We observed decreased expression of GS28 in rat ischemic hippocampus. In this study, we examined the role of GS28 and its molecular mechanisms in neuronal (SK-N-SH) cell death induced by hydrogen peroxide (H2O2). GS28 siRNA-transfected cells treated with H2O2 showed a significant increase in cytotoxicity under glutathione (GSH)-depleted conditions after pretreatment with buthionine sulfoximine, which corresponded to an increase of intracellular reactive oxygen species (ROS) in the cells. Pretreatment of GS28 siRNA-transfected cells with p38 chemical inhibitor significantly inhibited cytotoxicity; we also observed that p38 was activated in the cells by immunoblot analysis. We confirmed the role of p38 MAPK in cotransfected cells with GS28 siRNA and p38 siRNA in the cell viability assay, flow cytometry, and immunoblot. Involvement of apoptotic or autophagic processes in the cells was not shown in the cell viability, flow cytometry, and immunoblot analyses. However, pretreatment of the cells with necrostatin-1 completely inhibited H2O2-induced cytotoxicity, ROS generation, and p38 activation, indicating that the cell death is necroptotic. Collectively these data imply that H2O2 induces necroptotic cell death in the GS28 siRNA-transfected cells and that the necroptotic signals are mediated by sequential activations in RIP1/p38/ROS. Taken together, these results indicate that GS28 has a protective role in H2O2-induced necroptosis via inhibition of p38 MAPK in GSH-depleted neuronal cells.

White Matter Damage and Hippocampal Neurodegeneration Induced by Permanent Bilateral Occlusion of Common Carotid Artery in the Rat: Comparison between Wistar and Sprague-Dawley Strain

In order to reproduce chronic cerebral hypoperfusion as it occurs in human aging and Alzheimer's disease, we introduced permanent, bilateral occlusion of the common carotid arteries (BCCAO) in rats (Farkas et al, 2007). Here, we induced BCCAO in two different rat strains in order to determine whether there was a strain difference in the pathogenic response to BCCAO. Male Wistar and Sprague-Dawley (SD) rats (250-270 g) were subjected to BCCAO for three weeks. Kluver-Barrera and cresyl violet staining were used to evaluate white matter and gray matter damage, respectively. Wistar rats had a considerably higher mortality rate (four of 14 rats) as compared to SD rats (one of 15 rats) following BCCAO. Complete loss of pupillary light reflex occurred in all Wistar rats that survived, but loss of pupillary light reflex did not occur at all in SD rats. Moreover, BCCAO induced marked vacuolation in the optic tract of Wistar rats as compared to SD rats. In contrast, SD rats showed fewer CA1 hippocampal neurons than Wistar rats following BCCAO. These results suggest that the neuropathological process induced by BCCAO takes place in a region-specific pattern that varies according to the strain of rat involved.

Pre-ischemic Treatment with Ampicillin Reduces Neuronal Damage in the Mouse Hippocampus and Neostriatum after Transient Forebrain Ischemia

Ampicillin, a beta-lactam antibiotic, has been reported to induce astrocytic glutamate transporter-1 which plays a crucial role in protecting neurons against glutamate excitotoxicity. We investigated the effect of ampicillin on neuronal damage in the mouse hippocampus and neostriatum following transient global forebrain ischemia. Male C57BL/6 mice were anesthetized with halothane and subjected to bilateral occlusion of the common carotid artery for 40 min. Ampicillin was administered post-ischemically (for 3 days) and/or pre-ischemically (for 3~5 days until one day before the onset of ischemia). Pre- and post-ischemic treatment with ampicillin (50 mg/kg/day or 200 mg/kg/day) prevented ischemic neuronal death in the medial CA1 area of the hippocampus as well as the neostriatum in a dose-dependent manner. In addition, ischemic neuronal damage was reduced by pre-ischemic treatment with ampicillin (200 mg/kg/day). In summary, our results suggest that ampicillin plays a functional role as a chemical preconditioning agent that protects hippocampal neurons from ischemic insult.

A Case of Primary Paraganglioma that Arose in the Pancreas: the Color Doppler Ultrasonography and Dynamic CT Features

Paragangliomas rarely originate from the pancreas and they are characterized on imaging studies as well-marginated, hypervascular masses with cystic areas. We herein report on a case report of pancreatic paraganglioma in a 57-year-old woman, which was confirmed on pathology. Color Doppler ultrasonography and dynamic CT demonstrated a well-demarcated, extremely hypervascular mass with prominent intratumoral vessels and early contrast filling of the draining veins from the mass. Endoscopic retrograde cholangiopancreatography showed that the main pancreatic duct was displaced and mildly dilated.

Ischemic preconditioning in the rat hippocampus increases antioxidant activities but does not affect the level of hydroxyl radicals during subsequent severe ischemia

Several studies have demonstrated that ischemic preconditioning increases superoxide dismutase activity, but it is unclear how ischemic preconditioning affects events downstream of hydrogen peroxide production during subsequent severe ischemia and reperfusion in the hippocampus. To answer this question, we investigated whether ischemic preconditioning in the hippocampal CA1 region increases the activities of antioxidant enzymes glutathione peroxidase and catalase, resulting in a decrease in the level of hydroxyl radicals during subsequent severe ischemia-reperfusion. Transient forebrain ischemia was induced by four-vessel occlusion in rats. Ischemic preconditioning for 3 min or a sham operation was performed and a 15-min severe ischemia was induced three days later. Ischemic preconditioning preserved the CA1 hippocampal neurons following severe ischemia. The concentration of 2,3-dihydroxybenzoic acid, an indicator of hydroxyl radical, was measured using in vivo microdialysis technique combined with HPLC. The ischemia-induced increase in the ratio of 2,3-dihydroxybenzoic acid concentration relative to baseline did not differ significantly between preconditioned and control groups. On the other hand, activities of the antioxidant enzymes glutathione peroxidase-1 and catalase were significantly increased at 3 days after ischemic preconditioning in the hippocampus. Our results suggest that, in preconditioned rats, while hydrogen peroxide is generated from severe ischemia, the activity of catalase and glutathione peroxidase-1 is correspondingly increased to eliminate the excessive hydrogen peroxide. However, our results show that the enhanced activity of these antioxidant enzymes in preconditioned rats is not sufficient to decrease hydroxyl radical levels during subsequent severe ischemia-reperfusion.

Once-daily Dosing of Arbekacin Can Suppress the Formation of Small Colony Variants of Methicillin Resistant Staphylococcus aureus in an in vitro Pharmacodynamic Infection Model

BACKGROUND: Small colony variants (SCVs) of Staphylococcus aureus have emerged to be commonly associated with persistent and relapsing infections. Arbekacin (ABK) is one of a few alternatives to vancomycin in intractable case of methicillin resistant S. aureus (MRSA) infection. However, it has not yet been defined whethter ABK tends to be efficacious to the MRSA SCVs. In this study, we employed an in vitro pharmacodynamic infection model (IVPDIM) to define efficacies of ABK against MRSA SCVs. MATERIALS AND METHODS: Using four strains of clinically isolated MRSA (MRSA122, MRSA160, MRSA18, MRSA123), we adopted IVPDIM comprised of two-compartment in which effective surface-to-volume ratio of 5.34 cm(-1). Human pharmacokinetic regimen simulations of ABK were as follows: 100 mg every 12 h (q12h), 200 mg q24h, 200 mg q12h, and 400 mg q24h. Samples were taken from each model at 0, 1, 2, 4, 6, 12, 24, and 30 h, and the bacterial colony counts were determined. The experiments were repeated twice with ABK-administered groups and control group. RESULTS: MICs of ABK for MRSA122, MRSA160, MRSA18, and MRSA123 were 2, 2, 2, and 1 microgram/mL, respectively. In case of MRSA122, MRSA160, MRSA18, C(max)/MIC were less than 9.0 except for ABK 400 mg q24h regimen. In MRSA123, C(max)/MIC were 8.9 on average at ABK 100 mg q12h regimen. But, other regimen showed C(max)/MIC >9. Four regimens for 4 strains showed statistically different colony counts at 30 h (P=0.000). The more dosage or less frequent dosing interval, the more colonies tended to reduce in all strains. In 100 mg q12h groups, SCVs were observed in all strains within 24 h. With increment of dosage or changing dosing interval from q12h to 24h, SCVs were reduced (P=0.000). Regimen of 400 mg q24h did not let SCVs appear in all strains of MIC 2 microgram/mL during the experiments. CONCLUSION: SCVs were observed when MIC of ABK against MRSA were 1-2 microgram/mL, especially in most cases of C(max)/MIC <9. Those findings were also associated with re-growth of colony during the experiments. Once-daily dosing of ABK could reduce or eliminate the appearance of SCV.

Once-daily Dosing of Arbekacin Can Suppress the Formation of Small Colony Variants of Methicillin Resistant Staphylococcus aureus in an in vitro Pharmacodynamic Infection Model

BACKGROUND: Small colony variants (SCVs) of Staphylococcus aureus have emerged to be commonly associated with persistent and relapsing infections. Arbekacin (ABK) is one of a few alternatives to vancomycin in intractable case of methicillin resistant S. aureus (MRSA) infection. However, it has not yet been defined whethter ABK tends to be efficacious to the MRSA SCVs. In this study, we employed an in vitro pharmacodynamic infection model (IVPDIM) to define efficacies of ABK against MRSA SCVs. MATERIALS AND METHODS: Using four strains of clinically isolated MRSA (MRSA122, MRSA160, MRSA18, MRSA123), we adopted IVPDIM comprised of two-compartment in which effective surface-to-volume ratio of 5.34 cm(-1). Human pharmacokinetic regimen simulations of ABK were as follows: 100 mg every 12 h (q12h), 200 mg q24h, 200 mg q12h, and 400 mg q24h. Samples were taken from each model at 0, 1, 2, 4, 6, 12, 24, and 30 h, and the bacterial colony counts were determined. The experiments were repeated twice with ABK-administered groups and control group. RESULTS: MICs of ABK for MRSA122, MRSA160, MRSA18, and MRSA123 were 2, 2, 2, and 1 microgram/mL, respectively. In case of MRSA122, MRSA160, MRSA18, C(max)/MIC were less than 9.0 except for ABK 400 mg q24h regimen. In MRSA123, C(max)/MIC were 8.9 on average at ABK 100 mg q12h regimen. But, other regimen showed C(max)/MIC >9. Four regimens for 4 strains showed statistically different colony counts at 30 h (P=0.000). The more dosage or less frequent dosing interval, the more colonies tended to reduce in all strains. In 100 mg q12h groups, SCVs were observed in all strains within 24 h. With increment of dosage or changing dosing interval from q12h to 24h, SCVs were reduced (P=0.000). Regimen of 400 mg q24h did not let SCVs appear in all strains of MIC 2 microgram/mL during the experiments. CONCLUSION: SCVs were observed when MIC of ABK against MRSA were 1-2 microgram/mL, especially in most cases of C(max)/MIC <9. Those findings were also associated with re-growth of colony during the experiments. Once-daily dosing of ABK could reduce or eliminate the appearance of SCV.

A Simple Method for Predicting Hippocampal Neurodegeneration in a Mouse Model of Transient Global Forebrain Ischemia

In the present study, we developed a simple method to predict the neuronal cell death in the mouse hippocampus and striatum following transient global forebrain ischemia by evaluating both cerebral blood flow and the plasticity of the posterior communicating artery (PcomA). Male C57BL/6 mice were anesthetized with halothane and subjected to bilateral occlusion of the common carotid artery (BCCAO) for 30 min. The regional cerebral blood flow (rCBF) was measured by laser Doppler flowmetry. The plasticity of PcomA was visualized by intravascular perfusion of India ink solution. When animals had the residual cortical microperfusion less than 15% as well as the smaller PcomA whose diameter was less than one third compared with that of basilar artery, neuronal damage in the hippocampal subfields including CA1, CA2, and CA4, and in the striatum was consistently observed. Especially, when mice met these two criteria, marked neuronal damage was observed in CA2 subfield of the hippocampus. In contrast, after transient BCCAO, neuronal damage was consistently produced in the striatum, dependent more on the degree of rCBF reduction than on the plasticity of PcomA. The present study provided simple and highly reproducible criteria to induce the neuronal cell death in the vulnerable mice brain areas including the hippocampus and striatum after transient global forebrain ischemia.

Fluoxetine Modulates Corticostriatal Synaptic Transmission through Postsynaptic Mechanism

Fluoxetine, widely used for the treatment of depression, is known to be a selective serotonin reuptake inhibitor (SSRI), however, there are also reports that fluoxetine has direct effects on several receptors. Employing whole-cell patch clamp techniques in rat brain slice, we studied the effects of fluoxetine on corticostriatal synaptic transmission by measuring the change in spontaneous excitatory postsynaptic currents (sEPSC). Acute treatment of rat brain slice with fluoxetine (10microM) significantly decreased the amplitude of sEPSC (84.1+/-3.3%, n=7), but did not alter its frequency (99.1+/-4.7%, n=7). Serotonin (10microM) also significantly decreased the amplitude (81.2+/-3.9%, n=4) of sEPSC, but did not affect its frequency (105.8+/-8.0, n=4). The effect of fluoxetine was found to have the same trend as that of serotonin. We also found that the inhibitory effect of fluoxetine on sEPSC amplitude (93.0+/-1.9%, n=8) was significantly blocked, but not serotonin (84.3+/-1.6%, n=4), when the brain slice was incubated with p-chloroamphetamine (10microM), which depletes serotonin from the axon terminals and blocks its reuptake. These results suggest that fluoxetine inhibits corticostriatal synaptic transmission through postsynaptic, and that these effects are exerted through both serotonin dependent and independent mechanism.

NMDA Receptor-dependent Inhibition of Synaptic Transmission by Acute Ethanol Treatment in Rat Corticostriatal Slices

The effects of ethanol on corticostriatal synaptic transmission were examined, using extracellular recording and analysis of population spike amplitudes in rat brain slices, to study how acute ethanol intoxication impairs striatal function. Ethanol caused a decrease in population spike amplitudes in a dose dependent manner (50~200 mM). Pretreatment with picrotoxin, a gamma-amino butyric acid (GABA)A receptor antagonist, increased the population spikes but ethanol (100 mM) was still effective in decreasing the population spikes under this condition. In the presence of (DL)-2-amino-5-phosphonovaleric acid (APV), N-methyl-D-aspartate (NMDA) receptor antagonist, the inhibitory action of ethanol on population spikes was not shown. These results suggest that ethanol inhibits the glutamatergic corticostriatal synaptic transmission through blockade of NMDA receptors.

5-Hydroxytryptamine Inhibits Glutamatergic Synaptic Transmission in Rat Corticostriatal Brain Slice

Striatum is involved in the control of movement and habitual memory. It receives glutamatergic input from wide area of the cerebral cortex as well as an extensive serotonergic (5-hydroxytryptamine, 5-HT) input from the raphe nuclei. In our study, the effects of 5-HT on synaptic transmission were studied in the rat corticostriatal brain slice using in vitro whole-cell recording technique. 5-HT inhibited the amplitude as well as frequency of spontaneous excitatory postsynaptic currents (sEPSC) significantly, and neither gamma-aminobutyric acid (GABA) A receptor antagonist bicuculline (BIC), nor N-methyl-D-aspartate (NMDA) receptor antagonist, DL-2-amino-5-phosphonovaleric acid (AP-V) could block the effect of 5-HT. In the presence non-NMDA receptor antagonist, 2, 3-dioxo-6-nitro-1, 2, 3, 4-tetrahydrobenxo[f] quinoxaline-7-sulfonamide (NBQX), the inhibitory effect of 5-HT was blocked. We also figured out that 5-HT change the channel kinetics of the sEPSC. There was a significant increase in the rise time during the 5-HT application. Our results suggest that 5-HT has an effect on both pre- and postsynaptic site with decreasing neurotransmitter release probability of glutamate and decreasing the sensitivity to glutamate by increasing the rise time of non-NMDA receptor mediated synaptic transmission in the corticostriatal synapses.