Protective effect of topiramate on kainic acid–induced cell death in mice hippocampus

The protective effect of topiramate (TPM) on seizure-induced neuronal injury is well known; however, its molecular basis has yet to be elucidated. We investigated the effect and signaling mediators of TPM on seizure-induced hippocampal cell death in kainic acid (KA)-treated ICR mice. KA-induced hippocampal cell death was identified by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling. Immunoreactivity (IR) of p-Erk, p-Jnk, p-P38, and caspase-3, and caspase-3 activity were observed in the hippocampal region 3 h after KA (0.1 μg/5 μL, i.c.v.) administration, and/or TPM (100 mg/kg, i.p.) pretreatment. TPM attenuated seizure-induced neuronal cell death and reduced KA-induced p-Erk IR in the CA3 region of the hippocampus, but did not affect p-Jnk and p-P38. In addition, TPM reduced caspase-3 IR and activation by KA. KA-induced seizures were also suppressed by TPM pretreatment. TPM inhibits seizures, and decreases Erk phosphorylation and caspase-3 activation by KA, thereby contributing to protection from neuronal injury.     

NBQX or topiramate treatment after perinatal hypoxia-induced seizures prevents later increases in seizure-induced neuronal injury

PURPOSE: To evaluate the efficacy of NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f) quinoxaline-2,3-dione) and topiramate (TPM) given after hypoxia-induced seizures in preventing the delayed effect of hypoxia on subsequent susceptibility to seizures and neuronal injury. METHODS: We used "two-hit" rodent seizure model to study the long-term effect of perinatal hypoxia on later kainate (KA) seizure-induced neuronal damage and investigated the therapeutic efficacy of a postseizure treatment protocol in reversing the conditioning effect of early-life seizures. RESULTS: Hypoxia at P10 induces seizures without cell death but causes an increase in susceptibility to second seizures induced by KA as early as 96 h after hypoxia, and this lowered seizure threshold persists to adulthood. Furthermore, perinatal hypoxia increases KA-induced neuronal injury at postnatal day (P)21 and 28/30. Repeated doses of NBQX (20 mg/kg) or TPM (30 mg/kg) given for 48 h after hypoxia-induced seizures prevent the increase in susceptibility to KA seizure-induced hippocampal neuronal injury at P28/30. CONCLUSIONS: Our results suggest that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor blockade after hypoxia prevents the priming effect of perinatal hypoxia-induced seizures and that this protection occurs independent of its anticonvulsant action.     

Role of nicotinic acetylcholine receptors in the regulation of kainic acid-induced hippocampal cell death in mice

Kainic acid (KA) is a well-known excitatory, neurotoxic substance. In mice, morphological damage of hippocampus induced by KA administered intracerebroventricularly (i.c.v.) was markedly concentrated on the CA3 pyramidal neurons. In the present study, the possible role of nicotinic acetylcholine receptors (nAchRs) in hippocampal cell death induced by KA (0.1 microg) administered i.c.v. was examined. Methyllycaconitine (MC; nAchRs antagonist, 20 microg) attenuated KA-induced CA3 pyramidal cell death. KA increased immunoreactivities (IRs) of phorylated extracellular signal-regulated kinase (p-ERK; at 30 min), p-CaMK II (at 30 min), c-Fos (at 2 h), c-Jun (at 2 h), glial fibrillary acidic protein (GFAP at 1 day), and the complement receptor type 3 (OX-42; at 1 day) in hippocampal area. MC attenuated selectively KA-induced p-CaMK II, GFAP and OX-42 IR in the hippocampal CA3 region. Our results suggest that p-CaMK II may play as an important regulator responsible for the hippocampal cell death induced by KA administered i.c.v. in mice. Reactive astrocytes, which was meant by GFAP IR, and activated microglia, which was meant by OX-42 IR, may be a good indicator for measuring the cell death in hippocampal regions by KA-induced excitotoxicity. Furthermore, it is implicated that niconitic receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.     

Roles of adenosine receptors in the regulation of kainic acid-induced neurotoxic responses in mice

Kainic acid (KA) is a well-known excitatory and neurotoxic substance. In ICR mice, morphological damage of hippocampus induced by KA administered intracerebroventricularly (i.c.v.) was markedly concentrated on the hippocampal CA3 pyramidal neurons. In the present study, the possible role of adenosine receptors in hippocampal cell death induced by KA (0.1 microg) administered i.c.v. was examined. It has been shown that 3,7-dimethyl-1-propargylxanthine (DMPX; A2 adenosine receptors antagonist, 20 microg) reduced KA-induced CA3 pyramidal cell death. KA dramatically increased the phosphorylated extracellular signal-regulated kinase (p-ERK) immunoreactivities (IR) in dentate gyrus (DG) and mossy fibers. In addition, c-Jun, c-Fos, Fos-related antigen 1 (Fra-1) and Fos-related antigen 2 (Fra-2) protein levels were increased in hippocampal area in KA-injected mice. DMPX attenuated KA-induced p-ERK, c-Jun, Fra-1 and Fra-2 IR. However, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX; A1 adenosine receptor antagonist, 20 microg) did not affect KA-induced p-ERK, c-Jun, Fra-1 and Fra-2 IR. KA also increased the complement receptor type 3 (OX-42) IR in CA3 region of hippocampus. DMPX, but not PACPX, blocked KA-induced OX-42 IR. Our results suggest that p-ERK and c-Jun may function as important regulators responsible for the hippocampal cell death induced by KA administered i.c.v. in mice. Activated microglia, which was detected by OX-42 IR, may be related to phagocytosis of degenerated neuronal elements by KA excitotoxicity. Furthermore, it is implicated that A2, but not A1, adenosine receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.     

Effect of theophylline and trimethobenzamide when given during kainate-induced status epilepticus: an improved histopathologic rat model of human hippocampal sclerosis

PURPOSE: The most common pathology in temporal lobe epilepsy (TLE) is hippocampal sclerosis. It is controversial whether status epilepticus (SE) or prolonged seizures plus secondary cerebral injuries are pathogenic mechanisms of hippocampal sclerosis. This study addressed this question in rat models of TLE. METHODS: Hippocampal neuron densities and supragranular mossy fiber sprouting were determined in adult rats subjected to systemic kainate-induced SE (KA-only) and KA-induced SE followed 75 minutes later by theophylline (KA/Theo) or trimethobenzamide (KA/Tri). These drugs probably decrease seizure-induced cerebral hyperemia or hypertension. RESULTS: Compared with controls and KA-only rats, KA/Tri and KA/Theo rats showed decreased CA3b and CA1 neuron densities (i.e., greater Sommer's sector injury). In addition, KA/Tri rats showed that increased trimethobenzamide dosages were associated with decreased hilar, CA3c, CA3b, CA1, and subiculum neuron densities. There were no significant differences in supragranular mossy fiber sprouting between KA-only, KA/Tri, and KA/Theo rats. CONCLUSIONS: Pharmacologic manipulations during KA-induced SE are associated with differences in hippocampal pathology, especially in Sommer's sector, and the final pattern of damage and axon sprouting shows histopathologic similarities to that in patients with hippocampal sclerosis. Our findings support the hypothesis that secondary physiologic insults during SE that are likely to decrease seizure-induced cerebral hyperemia and hypertension may generate greater hippocampal neuronal injury compared with SE alone, and this may be a pathogenic mechanism of human hippocampal sclerosis in patients with TLE.     

Resistance of immature hippocampus to morphologic and physiologic alterations following status epilepticus or kindling.

Seizures in adult rats result in long-term deficits in learning and memory, as well as an enhanced susceptibility to further seizures. In contrast, fewer lasting changes have been found following seizures in rats younger than 20 days old. This age-dependency could be due to differing amounts of hippocampal neuronal damage produced by seizures at different ages. To determine if there is an early developmental resistance to seizure-induced hippocampal damage, we compared the effects of kainic acid (KA)-induced status epilepticus and amygdala kindling on hippocampal dentate gyrus anatomy and electrophysiology, in immature (16 day old) and adult rats. In adult rats, KA status epilepticus resulted in numerous silver-stained degenerating dentate hilar neurons, pyramidal cells in fields CA1 and CA3, and marked numerical reductions in CA3c pyramidal neuron counts (-57%) in separate rats. Two weeks following the last kindled seizure, some, but significantly less, CA3c pyramidal cell loss was observed (-26%). Both KA status epilepticus and kindling in duced mossy-fiber sprouting, as evidenced by ectopic Timm staining in supragranular layers of the dentate gyrus. In hippocampal slices from adult rats, paired-pulse stimulation of perforant path axons revealed a persistent enhancement of dentate granule-cell inhibition following KA status epilepticus or kindling. While seizures induced by KA or kindling in 16-day-old rats were typically more severe than in adults, the immature hippocampus exhibited markedly less KA-induced cell loss (-22%), no kindling-induced loss, no detectable synaptic rearrangement, and no change in dentate inhibition. These results demonstrate that, in immature rats, neither severe KA-induced seizures nor repeated kindled seizures produce the kind of hippocampal damage and changes associated with even less severe seizures in adults. The lesser magnitude of seizure-induced hippocampal alterations in immature rats may explain their greater resistance to long-term effects of seizures on neuronal function, as well as future seizure susceptibility. Conversely, hippocampal neuron loss and altered synaptic physiology in adults may contribute to increased sensitivity to epileptogenic stimuli, spontaneous seizures, and behavioral deficits.     

Neuroprotection of co-activation of GABA receptors by preventing caspase-3 denitrosylation in KA-induced seizures

Previous studies have demonstrated that kainic acid (KA)-induced seizures can cause the enhancement of excitation and lead to neuronal death in rat hippocampus. Co-activation of the inhibitory GABA receptors can attenuate the excitatory JNK3 apoptotic signaling pathway via inhibiting the increased assembly of the GluR6-PSD-95-MLK3 signaling module induced by KA in epileptic rat hippocampal CA1 and CA3 regions. Caspase-3 is a cysteine protease located in both the cytoplasm and mitochondrial intermembrane space that is a central effector of many apoptotic pathways. We designed experiments to elucidate the underlying molecular mechanisms of procaspase-3 activation and neuroprotection of co-activation of GABA receptors against neuronal death induced by KA. In this study, we show that co-activation of GABA receptors can attenuate the Fas/FasL apoptotic signaling pathway and inhibit the increased of thioredoxin reductase activity induced by KA, subsequently inhibit the activation of procaspase-3 by diminishing the denitrosylation of its active-site thiol and decreasing the cleavage of the caspase-3 zymogen to its active subunits. These results indicate that co-activation of GABA receptors results in neuroprotection by preventing caspase-3 denitrosylation in KA-induced seizure of rats.     

Kainic acid-induced seizures cause neuronal death in infant rats pretreated with lipopolysaccharide

A major controversy in human epilepsy is whether severe seizures in infants or young children cause brain damage and subsequent epilepsy. Kainic acid (KA) produces severe seizures in infant rats, but hippocampal neuronal death and mossy fibre sprouting have not been previously demonstrated. There are similarities between lipopolysaccharide (LPS) pretreatment and KA-induced seizures in rats and the febrile convulsion of young children, in that both processes are associated with an immune stimulus and seizures. Infant rats, co-treated with LPS and KA, showed hippocampal neuronal death and mossy fibre sprouting. Taken together, our results suggest that severe febrile convulsion of young children may cause hippocampal damage and synaptic reorganization.     

MRS Metabolic Markers of Seizures and Seizure-Induced Neuronal Damage

Summary: Purpose: Proton magnetic resonance spectroscopy (MRS) was used to identify specific in situ metabolic markers for seizures and seizure-induced neuronal damage. Kainic acid (KA)-induced seizures lead to histopathologic changes in rat brain. The protective effect of cycloheximide treatment against neuronal damage caused by KA-induced seizures was studied, using in situ proton MRS imaging technique.
Methods: Rats were pretreated with placebo or cycloheximide 1 h before KA injection. Rat brains (n = 25) were scanned at the level of the hippocampus before, during, and 24 h after seizures. Spectra were recorded and the relative ratios of N-acetylaspartate (NAA), choline (cho), and lactate (Lac) to creatine (Cr) were calculated and compared between groups.
Results: A significant increase in Lac ratios was observed in KA-treated rats during and 24 h after seizure onset and this increase was prevented by cycloheximide pretreatment. NAA ratios were significantly higher during the ictal phase following KA treatment and this effect was not affected by cycloheximide pretreatment. Nissl staining confirmed previously reported prevention of KA-induced neuronal loss in CA1 and CA areas of the hippocampus by cycloheximide pretreatment.
Conclusions: Our results suggest that in situ Lac increase is a marker of seizure-induced neuronal damage, whereas N-acetylaspartate (NAA) changes during and after status epilepticus may be a reflection of neuronal activity and damage, respectively.     

In vivo delivery of a XIAP (BIR3-RING) fusion protein containing the protein transduction domain protects against neuronal death induced by seizures

The prevention of cell apoptosis is a promising strategy for neuroprotection against brain injury in seizures. X-linked inhibitor of apoptosis protein (XIAP) is regarded as the most potent inhibitor of cell apoptosis. In the present study, we fused the protein transduction domain (PTD) of Antennapedia Homeodomain of Drosophila (AntpHD) to XIAP (BIR3-RING) and explored the neuroprotective effect of XIAP in rats with seizures induced by kainic acid (KA). KA triggered neuronal death in the ipsilateral CA3 subfield of the hippocampus and activation of caspase-3 and -9. PTD-XIAP fusion protein can be delivered into cos7 cells in vitro. We used intraperitoneal injection to deliver the PTD-XIAP fusion protein which can enter into brain, significantly decrease the TUNEL positive cells and increase the number of surviving cells in the ipsilateral CA3 subfield of the hippocampus at 24 h after KA-induced seizures. Furthermore, PTD-XIAP fusion protein attenuated activated caspase-3 and -9. These results demonstrate the neuroprotective effect of PTD-XIAP fusion protein against brain injury possibly through the inhibition of caspase. The significance of these findings in the treatment of epilepsy still needs to be extensively studied.     

Cycloheximide inhibits neurotoxic responses induced by kainic acid in mice

In the present study, we examined the effect of cycloheximide on various pharmacological responses induced by kainic acid (KA) administered intracerebroventricularly (i.c.v.) in mice. In a passive avoidance test, a 20-min cycloheximide (200mg/kg, i.p.) pretreatment prevented the memory impairment induced by KA. The morphological damage induced by KA (0.1microg) in the hippocampus was markedly concentrated in the CA3 pyramidal neurons and cycloheximide effectively prevented the KA-induced pyramidal cell death in CA3 hippocampal region. In immunohistochemical study, KA dramatically increased the phosphorylation of extracellular signal-regulated protein kinase (p-ERK), c-Jun N-terminal kinase 1 (p-JNK1), and calcium/calmodulin-dependent protein kinase II (p-CaMK II). Cycloheximide attenuated the increased p-ERK, p-JNK1, and p-CaMK II levels induced by KA. Furthermore, cycloheximide inhibited the increased c-Fos and c-Jun protein expression levels induced by KA in the hippocampus. The activation of microglia was detected in KA-induced CA3 cell death region by immunostaining with a monoclonal antibody against the OX-42. Cycloheximide inhibited KA-induced increase of OX-42 immunoreactivity. Our results suggest that the increased expression of the c-Fos, c-Jun, and phosphorylation of ERK, JNK1, and CaMK II proteins may play important roles in the memory impairment and the cell death in CA3 region of the hippocampus induced by i.c.v. KA administration in mice. Furthermore, the activated microglia may be related to phagocytosis of degenerated neuronal elements induced by KA.     

Characterization of neuronal death induced by focally evoked limbic seizures in the C57BL/6 mouse

Research into the molecular mechanisms of epileptic brain injury is hampered by the resistance of key mouse strains to seizure-induced neuronal death evoked by systemically administered excitotoxins such as kainic acid. Because C57BL/6 mice are extensively employed as the genetic background for transgenic/knockout modeling in cell death research but are seizure resistant, we sought to develop a seizure model in this strain characterized by injury to the hippocampal CA subfields. Adult male C57BL/6 mice underwent focally evoked seizures induced by intraamygdala microinjection of kainic acid. Kainic acid (KA) effectively elicited ipsilateral CA3 pyramidal neuronal death within a narrow dose range of 0.1-0.3 microg, with mortality < 10%. With employment of the most consistent (0.3 microg) dose, seizures were terminated 15, 30, 60, or 90 min after KA by diazepam. Damage was largely restricted to the ipsilateral CA3 subfield of the hippocampus, but injury was also consistent within CA1, suggesting that this mouse model better reflects the hippocampal neuropathology of human temporal lobe epilepsy than does the rat, in which CA1 is typically spared. Confirming this CA1 injury as seizure specific and not a consequence of ischemia, we used laser-Doppler flowmetry to determine that cerebral perfusion did not significantly change (97% to 118%) over control. Degenerating cells were > 95% neuronal as determined by neuron-specific nuclear protein (NeuN) counterstaining of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeled (TUNEL) brain sections. Furthermore, TUNEL-positive cells often exhibited the morphological features of apoptosis, and small numbers were positive for cleaved caspase-3. These data establish a mouse model of focally evoked seizures in the C57BL/6 strain associated with a restricted pattern of apoptotic neurodegeneration within the hippocampal subfields that may be applied to research into the molecular basis of neuronal death after seizures.     

The protective effect of a ketogenic diet on kainic acid-induced hippocampal cell death in the male ICR mice

This study was designed to evaluate the antiapoptotic effects of a ketogenic diet (KD) through histological (cresyl violet staining, TUNEL staining and immunohistochemistry) and behavioral studies using kainic acid (KA, 25mg/kg i.p.)-induced seizures in male ICR mice. KA-induced seizure in rodents is widely used as an experimental model for human temporal lobe epilepsy because of their behavioral and pathological similarities. A KA-induced seizure causes neuronal damage in hippocampal pyramidal neurons and involves a caspase-3-mediated apoptotic pathway. In this study, the seizure onset time of the KD-fed group was delayed compared to that of the group fed a normal diet (ND) after a systemic KA injection. Histological studies revealed that KA caused pyknosis in most of the hippocampal areas in the ND-fed group, however, well-preserved pyramidal neurons were detected in the hippocampus of mice that had been on KD for 1 month, which began on postnatal day 21. The number of TUNEL-positive cells and caspase-3-positive cells in the hippocampus of the KD-fed group was lower than that of the ND-fed group. These findings indicate that KD has an antiepileptic effect via a neuroprotective action that involves the inhibition of caspase-3-mediated apoptosis of hippocampal neurons.     

Inhibition of delayed induction of p38 mitogen-activated protein kinase attenuates kainic acid-induced neuronal loss in the hippocampus

The activation of p38 mitogen-activated protein kinase (MAPK) has been implicated in the pathological changes accompanying inflammatory and apoptotic processes of various cell types including neurons. In a kainic acid (KA)-induced mouse seizure model, p38 MAPK is induced in reactive astrocytes in the CA3 region of the hippocampus where severe neuronal loss occurs. Here we report the delayed and protracted activation of p38 MAPK in the CA3 region of the hippocampus of mice treated with KA. In this model, the inhibition of p38 MAPK isoforms by SB203580, a specific inhibitor, attenuated neuronal loss in the CA3 and CA1 regions of the hippocampus, which was accompanied by the suppression of the p38 MAPK activation as well as astrogliosis. Thus, the delayed and sustained induction of p38 MAPK plays a crucial role in the neuronal damage of KA-induced brain seizures.     

Dentate granule cells are essential for kainic acid-induced wet dog shakes but not for seizures

The purpose of this study was to determine the role that dentate granule cells play in wet dog shakes (WDS), behavioral seizures, and hippocampal cell loss caused by systemic administration of kainic acid (KA). Rats were given bilateral injections of colchicine (COL) into the hippocampal formation to selectively lesion dentate granule cells. Two weeks later, they were injected subcutaneously with KA and were observed for WDS and seizures. Seizures were terminated with pentobarbital 2.5 hr after KA injection, and the rats were killed 48 hr later. The integrity of hippocampal cell populations and projections to the hippocampal formation from entorhinal cortex was assessed with radioimmunoassay and immunostaining for methionine-enkephalin (ME) and dynorphin (DYN) A, as well as with Timm and Nissl staining. Results indicate that COL injections eliminated KA-induced WDS, did not affect the latency to onset of seizures, and potentiated KA-induced cell loss in the CA3 region of hippocampus. COL lesions eliminated ME and DYN immunostaining of granule cells, but not ME immunostaining of entorhinal afferents to the dentate gyrus or Ammon's horn. These findings indicate that granule cells are an essential neuronal link in the expression of KA-induced WDS, but that seizures propagate along other pathways in the limbic system.     

Ketogenic diet reduces Smac/Diablo and cytochrome c release and attenuates neuronal death in a mouse model of limbic epilepsy

The ketogenic diet (KD) is effective in the treatment of refractory epilepsy, yet the molecular mechanisms underlying its antiepileptic effects have not been determined. There is increasing evidence that neuronal cell death induced by seizures via mitochondrial pathway and seizures can lead to mitochondrial release of cytochrome c, and we have shown previously that translocation of Smac/DIABLO into the cytosol play a role in the brain damage in a model of limbic seizure. In the present study, we explored the neuroprotective effect of KD in C57BL/6 mice with seizures induced by kainic acid (KA). Status epilepticus triggered by intra-amygdaloid microinjection of KA lead to neuronal death in the selective ipsilateral CA3 subfield of the hippocampus and mitochondrial release of Smac/DIABLO and cytochrome c. We found that KD significantly decreased neuronal death in the ipsilateral CA3 at 24h after KA-induced seizures. Furthermore, KD reduced Smac/DIABLO and cytochrome c release from mitochondria, attenuated activation of casepase-9 and caspase-3 following seizures. These results demonstrate that the neuroprotective effect of KD against brain injury induced by limbic seizures, at least partially, is associated with inhibition of mitochondrial release of Smac/DIABLO and cytochrome c.     

Effects of nitric oxide synthase inhibition on the cerebral circulation and brain damage during kainic acid-induced seizures in newborn rabbits.

The nitric oxide (NO) synthase inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME), was used to investigate the effect of endogenous NO on the cerebral circulation and brain damage during kainic acid (KA)-induced seizures in newborn rabbits. The cerebral blood flow (CBF), by laser doppler flowmetry, cerebral oxygenation (concentrations of oxy-(HbO2), deoxy-(HbR) and total hemoglobin (tHb) in brain tissue), by near-infrared spectroscopy (NIRS), mean arterial blood pressure (MABP), electroencephalography (EEG), and hippocampal neuronal damage were evaluated. Pretreatment with L-NAME caused significant decreases in CBF, HbO2, and tHb, and a significant increase in HbR during KA-induced seizures, compared with pretreatment with saline (P < 0.05), without a significant difference in MABP. Our study also demonstrated that pretreatment with L-NAME reduced the seizure activity and neuronal cell death in the hippocampus elicited by the systemic administration of KA in the neonatal brain. These results suggest that NO is of major importance in the neurodestructive process in spite of its roles in maintaining both the CBF and cerebral oxygenation during KA-induced seizures in the neonatal brain.     

Kainic Acid-Induced Seizures Modulate Akt (SER473) Phosphorylation in the Hippocampus of Dopamine D2 Receptor Knockout Mice

Dopamine D2 receptor (D2R) signalling has been shown to modulate seizure-induced hippocampal cell death. D2R knockout (D2R?/?) mice are more susceptible to kainic acid (KA)-induced excitotoxicity, displaying cell death in the CA3 subfield of the hippocampus at KA doses not damaging in wild-type (WT) animals. Absence of D2R signalling in the hippocampus leads to activation (dephosphorylation) of glycogen synthase kinase 3β (GSK-3β) after KA (20 mg/kg), which is not associated with a change in the phosphorylation of the GSK-3β regulator Akt at the canonical threonine 308 residue. In the present study, we investigated alternative pathways responsible for the activation of GSK-3β in the hippocampus of the D2R?/? mice 24 h following KA-induced seizures. Here, we show that phosphorylation of Akt occurs at serine 473 (Ser473) in the CA3 region of WT but not D2R?/? mice following KA. Moreover, the CA1 subregion, which does not undergo neurodegeneration in either WT or D2R?/? mice, displays a strong induction of Akt (Ser473) phosphorylation after KA. Additionally, the vulnerability in the CA3 is not associated with changes to p38MAPK and Dishevelled activation, and β-catenin does not appear to be a downstream target of the GSK-3β. Thus, we propose that GSK-3β phosphorylation-mediated hippocampal cell survival may depend on Akt (Ser473) phosphorylation; loss of D2R-mediated signalling in the CA3 region of D2R?/? mice leads to reduced Akt (Ser473) phosphorylation rendering neurons more vulnerable to apoptosis. Further investigation is required to fully elucidate the GSK-3β targets involved in D2R-dependent response to excitotoxicity.     

Effect of the calcineurin inhibitor FK506 on K+–Cl? cotransporter 2 expression in the mouse hippocampus after kainic acid-induced status epilepticus

Calcineurin (CaN)-mediated excitotoxicity impairs γ-aminobutyric acid (GABA) transmission and induces neuronal apoptosis. Ca(2+)-dependent K(+)-Cl(-) cotransporter 2 (KCC2) participates in GABAergic inhibitory transmission. However, the mechanism by which CaN mediates GABA receptor-mediated KCC2 in seizures is not fully understood. In the present study, we investigated the altered expression of KCC2 and the effects of the CaN inhibitor FK506 on KCC2 expression in the mouse hippocampus following kainic acid (KA) treatment. FK506 was injected twice 24 h and 30 min before KA treatment and then mice were treated with KA and killed 2 days later. FK506 had anticonvulsant effect on KA-induced seizure activities. CaN cleavage was evident in the hippocampus 24 h after KA treatment. FK506 pretreatment blocked the truncation of CaN in the KA-treated hippocampus. Cresyl violet and TUNEL staining showed that FK506 prevented KA-induced hippocampal cell death. In particular, Western blot analysis showed that KCC2 expression was time dependent, with a peak at 6 h and a return to decreased levels at 48 h, whereas FK506 pretreatment inhibited the KA-induced decrease in KCC2 expression in the hippocampus. Immunofluorescence showed that FK506 pretreatment protected the loss of inhibitory GABAergic KCC2-expressing neurons following KA treatment. Taken together, these results provide evidence that altered KCC2 expression may be associated with Ca(2+)-mediated seizure activity and indicate that neuron-specific KCC2 may be involved in neuroprotection after seizures.