Kainic Acid Treatment Research Articles

Decreased interaction between FoxO3a and Akt correlates with seizure-induced neuronal death

Status epilepticus (SE) leads to neurodegeneration which likely contributes to the development of chronic temporal lobe epilepsy (TLE). Therefore, neuroprotection following SE is considered as a promising strategy for preventing chronic TLE, but molecular changes that occur following SE still remain unclear. The Forkhead homeobox type O (FoxO) family of Forkhead transcription factors mediates cell death in several pathological conditions, but the role of FoxO in the excitotoxic effects of kainic acid (KA) remains largely unknown. The present study examined how FoxO3a and its interaction with other proteins changed in response to excitotoxic stimuli in the mouse hippocampus after SE. Mice were given intraperitoneal injection of kainate and seizure behavior was monitored for 2h to ensure SE. Western blot analyses, co-immunoprecipitation experiments, sub-cellular fractionation and double immunofluorescence analyses were used to determine changes in levels of FoxO3a, Akt, Bim, cleaved caspase-3 and phospho-FoxO3a or phospho-Akt, and their interactions at 6 or 24h after KA treatment. We found that SE activated FoxO3a and increased levels of Bim or cleaved caspase-3, and decreased levels of phospho-FoxO3a or phospho-Akt in the hippocampus. In addition, we noted extensive hippocampal cell death at 24h after KA treatment, evidenced by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), fluoro-jade B or anti-active caspase-3 staining. Furthermore, co-immunoprecipitation experiments revealed that phospho-Akt interaction with FoxO3a was significantly lowered in the hippocampus at 24h after KA treatment, paralleling enhanced Bim levels and Bim interaction with Bcl-xL. Moreover, double immunofluorescence analyses showed increased co-localization of FoxO3a or Bim and TUNEL in the hippocampi at 24h after KA treatment. Identifying molecular mechanism underlying SE-induced neuronal death can provide a novel strategy to protect against seizure-induced neuronal injury. We found that Akt-FoxO3a signaling relates to seizure-induced neuronal death, providing insight into neuroprotection following SE.

Effects of FK506 on long-term potentiation observed by optical imaging in organotypic hippocampal slice culture

Electrophysiological and biochemical assays were used to observe long-term changes in synaptic efficacy following electrical and/or pharmacological manipulation of synaptic function. We investigated the effects of FK506 on synaptic plasticity in organotypic hippocampal slice culture (OHSC) using optical imaging system. Hippocampal slices were prepared from 6-7 day-old rats using a tissue chopper and placed on a membrane insert. 18 hr after kainic acid (KA) treatment, significant delayed neuronal death which was quantified by propidium iodide, NeuN, and TUNEL staining was detected in CA3 area at 24 hr after KA treatment. The neuronal death was prevented significantly at 24 hr after 0.1 μM FK506 treatment. In the electrophysiology study, optical signals were observed by long-term potentiation (LTP) followed by the stimulation of the Schaffer collateral pathway. The improvement in amount of LTP was found in the FK506-treated group. These results suggest that FK506 may have a beneficial role in recovery of synaptic efficacy following KA-induced neuronal injury.

Loss of hippocampal neurons after kainate treatment correlates with behavioral deficits.

Treating rats with kainic acid induces status epilepticus (SE) and leads to the development of behavioral deficits and spontaneous recurrent seizures later in life. However, in a subset of rats, kainic acid treatment does not induce overt behaviorally obvious acute SE. The goal of this study was to compare the neuroanatomical and behavioral changes induced by kainate in rats that developed convulsive SE to those who did not. Adult male Wistar rats were treated with kainic acid and tested behaviorally 5 months later. Rats that had experienced convulsive SE showed impaired performance on the spatial water maze and passive avoidance tasks, and on the context and tone retention tests following fear conditioning. In addition, they exhibited less anxiety-like behaviors than controls on the open-field and elevated plus-maze tests. Histologically, convulsive SE was associated with marked neuron loss in the hippocampal CA3 and CA1 fields, and in the dentate hilus. Rats that had not experienced convulsive SE after kainate treatment showed less severe, but significant impairments on the spatial water maze and passive avoidance tasks. These rats had fewer neurons than control rats in the dentate hilus, but not in the hippocampal CA3 and CA1 fields. Correlational analyses revealed significant relationships between spatial memory indices of rats and neuronal numbers in the dentate hilus and CA3 pyramidal field. These results show that a part of the animals that do not display intense behavioral seizures (convulsive SE) immediately after an epileptogenic treatment, later in life, they may still have noticeable structural and functional changes in the brain.

Distinguishing hair cell from neural potentials recorded at the round window

Almost all patients who receive cochlear implants have some acoustic hearing prior to surgery. Electrocochleography (ECoG), or electrophysiological measures of cochlear response to sound, can identify remaining auditory nerve activity that is the basis for this residual hearing and can record potentials from hair cells that are no longer functionally connected to nerve fibers. The ECoG signal is therefore complex, being composed of both hair cell and neural signals. To identify signatures of different sources in the recorded potentials, we collected ECoG data across frequency and intensity from the round window of gerbils before and after treatment with kainic acid, a neurotoxin. Distortions in the recorded waveforms were produced by different sources over different ranges of frequency and intensity. In response to tones at low frequencies and low-to-moderate intensities, the major source of distortion was from neural phase-locking that was sensitive to kainic acid. At high intensities at all frequencies, the distortion was not sensitive to kainic acid and was consistent with asymmetric saturation of the hair cell transducer current. In addition to loss of phase-locking, changes in the envelope after kainic acid treatment indicate that sustained neural firing combines with receptor potentials from hair cells to produce the envelope of the response to tones. These results provide baseline data to interpret comparable recordings from human cochlear implant recipients.

A comparison of frailty of primary neurons, embryonic, and aging mouse cortical layers

Superficial layers I to III of the human cerebral cortex are more vulnerable toward Aβ peptides than deep layers V to VI in aging. Three models of layers were used to investigate this pattern of frailty. First, primary neurons from E14 and E17 embryonic murine cortices, corresponding respectively to future deep and superficial layers, were treated either with Aβ1–42, okadaic acid, or kainic acid. Second, whole E14 and E17 embryonic cortices, and third, in vitro separated deep and superficial layers of young and old C57BL/6J mice, were treated identically. We observed that E14 and E17 neurons in culture were prone to death after the Aβ and particularly the kainic acid treatment. This was also the case for the superficial layers of the aged cortex, but not for the embryonic, the young cortex, and the deep layers of the aged cortex. Thus, the aged superficial layers appeared to be preferentially vulnerable against Aβ and kainic acid. This pattern of vulnerability corresponds to enhanced accumulation of senile plaques in the superficial cortical layers with aging and Alzheimer's disease.

Possible Protecting Role of TNF-α in Kainic Acid-induced Neurotoxicity Via Down-Regulation of NFκB Signaling Pathway

We have shown previously, that mice lacking tumor necrosis factor-α (TNF-α) receptor 1 (TNFR1) exhibit greater hippocampal neurodegeneration, suggesting that TNFR1 may be protective in kainic acid (KA)-induced neurotoxicity. Here, we aim to clarify the role of TNF-α in neurodegenerative disorders and to elucidate its potential signaling pathways. TNF-α knockout (KO) mice and wild-type (WT) mice were treated with KA intranasally and, seizure severity measures obtained, Behavioral tests, including Elevated Plus-Maze™, open-field, Y-maze were also performed. Five days following KA treatment, immunohistochemical methods were used to assess neuronal degeneration and glial activation. The production of nitric oxide (NO) and the expression of nuclear factor kappaB (NF-κB) and AKT in the hippocampus were also measured. Compared with WT mice, TNF-α KO mice were more susceptible to KA-induced neurotoxicity, as demonstrated by more severe seizures, measurable behavior changes, greater neuronal degeneration in hippocampus, elevated glial activation and NO production. Additionally, KA-treatment up-regulated the expression of NFκB in TNF-α KO mice to a greater degree than in KA-treated WT mice. We conclude that TNF-α deficiency adversely influences KA-induced neurotoxicity and that TNF-α may play a protective role in KA-induced neurotoxicity via the down-regulation of NFκB signaling pathway.

Hyponatremia augments kainic-acid induced status epilepticus in the mouse: A model for dysmetabolic status epilepticus

Status epilepticus (SE) is a dreaded neurological emergency. A reignited interest in SE has resulted in a more adaptive use of treatment protocols. More knowledge on SE of various aetiologies is therefore needed. We are interested in treatment of SE under hyponatremia, and have here evaluated whether SE induced by systemic kainic acid could be a suitable platform for such studies. Acute hyponatremia was induced in C57/BL6 mice by intraperitoneal injection of dDAVP and water loading. Hyponatremic mice displayed an increased frequency of epileptiform spikes on EEG and 5/9 hyponatremic mice displayed electrographic seizures. After kainic acid (20mg/kg) treatment, hyponatremic mice displayed significantly longer time with electrographic seizure activity, which was also seen after treatment with diazepam (20mg/kg). We conclude that hyponatremia augments kainic acid-induced SE, This model might be a valuable platform for studies on treatment of SE in hyponatremia.

Lactation Reduces Glial Activation Induced by Excitotoxicity in the Rat Hippocampus

Motherhood induces a series of adaptations in the physiology of the female, including an increase of maternal brain plasticity and a reduction of cell damage in the hippocampus caused by kainic acid (KA) excitotoxicity. We analysed the role of lactation in glial activation in the hippocampal fields of virgin and lactating rats after i.c.v. application of 100ng of KA. Immunohistochemical analysis for glial fibrillary acidic protein (GFAP) and ionised calcium binding adaptor molecule 1 (Iba-1), which are markers for astrocytes and microglial cell-surface proteins, respectively, revealed differential cellular responses to KA in lactating and virgin rats. A significant astrocyte and microglial response in hippocampal areas of virgin rats was observed 24h and 72h after KA. By contrast, no increase in either GFAP- or Iba-1-positive cells was observed in response to KA in the hippocampus of lactating rats. Western blot analysis of GFAP showed an initial decrease at 24h after KA treatment, with an increase at 72h in the whole hippocampus of virgin but not of lactating rats. The number of GFAP-positive cells was increased by lactation in the dentate gyrus of the hippocampus but not in CA1 and CA3 areas. The present results indicate that lactating rats exhibit diminished responses of astrocyte and microglial cells in the hippocampus to damage induced by KA, supporting the notion that the maternal hippocampus is resistant to excitotoxic insults.

Excitotoxicity-induced immediate surge in hippocampal prostanoid production has latent effects that promote chronic progressive neuronal death

Excitotoxicity is involved in neurodegenerative conditions. We investigated the pathological significance of a surge in prostaglandin production immediately after kainic acid (KA) administration [initial phase], followed by a sustained moderate elevation in prostaglandin level [late phase] in the hippocampus of juvenile rats. Numerous pyknotic hippocampal neurons were observed 72h after KA treatment; this number remained elevated on days 10 and 30. Gross hippocampal atrophy was observed on days 10 and 30. Pre-treatment with indomethacin ameliorated neuronal death on days 10 and 30, and prevented hippocampal atrophy on day 30. Microglial response was moderated by the indomethacin pre-treatment. Blockade of only late-phase prostaglandin production by post-treatment with indomethacin ameliorated neuronal death on day 30. These findings suggest a role for initial-phase prostaglandin production in chronic progressive neuronal death, which is exacerbated by late-phase prostaglandin production. Blockade of prostaglandin production has therapeutic implications in preventing long-term neurological sequelae following excitotoxic brain damage.

Kainic acid-induced seizure activity alters the mRNA expression and G-protein activation of the opioid/nociceptin receptors in the rat brain cortex

The opioid/nociceptin receptors are involved in many neurological disorders such as Alzheimer's disease, Parkinson's disease and epilepsy. Kainic acid (KA) is an analog of the excitatory amino acid transmitter glutamate and the systemic administration of KA induces status epilepticus (SE) in rodents. In this study, we examined the alterations in the G-protein activity and the gene expression levels of mu, kappa, delta opioid and nociceptin receptors (MOPr, KOPr, DOPr and NOPr) as well as PNOC, the precursor polypeptide of nociceptin-OFQ (N/OFQ) in KA-induced seizures in the rat brain cortex. KA was used to create seizures with the dose of 10 mg/kg body weight i.p. Following the KA administration, the rats were observed for 3 h to assess seizure activity. Seizures occurred approximately 45 min after the KA injection. Only rats exhibiting full limbic seizures, forelimb clonus with rearing, were used in this study. All animals were decapitated 4 h after the administration of KA. Our [(35)S]GTPγS binding results showed that there was a significant difference in both the affinity and efficacy particularly one of NOPr stimulation following KA treatment. Slight, but significant increase was observed for MOPr. Moreover PNOC, NOPr and MOPr mRNA levels were increased by KA treatment but there were no significant changes in the levels of DOPr and KOPr mRNAs. These results show that the activities of opioid/nociceptin receptors can be modified by KA-treatment, and MOPr, PNOC and NOPr are the most responsive to KA-induced seizures in the rat brain cortex.

Protective Mechanism of Melatonin on Kainic Acid Induced Immune Modulatory Effect on Lymphocytes Derived from Mouse Spleen

Toxicity occurs when cells develop inflammation due to exposure to several toxic substances. The study was aimed to assess the damage (toxicity) induced by kainic acid on splenic lymphocyte and enriched T lymphocyte under in vitro conditions and the protective role of exogenous melatonin against this damage (toxicity). The study included assessment of inflammatory mechanism by the expression of immune modulatory cytokine mediators using real time PCR. Oxidative stress (reactive oxygen species) and nitrosative stress (reactive nitrogen species) were also studied to determine the free radical production. Interestingly, kainic acid caused severe splenic lymphocytes and enriched T lymphocyte damage which was evident from deleterious alterations in various parameters. Kainic acid treatment (1 mM) resulted in increased mRNA expression of cytokines like tumour necrosis factor-beta, interleukin 6, interleukin 1, interferon gamma, mitogen-activated protein kinase gene-14, inducible nitric oxide synthase and decreased interleukin 10 mRNA expression. Tritiated (3H) Thymidine Incorporation study signifies that kainic acid treatment (1 mM) increased the proliferation of splenic and enriched T lymphocytes. These changes were normalized by exogenous administration of melatonin (0.25-1.0 mM) in combination with kainic acid. Flow Cytometry Analysis using Annexin V apoptosis assay kit revealed an increase in apoptotic and necrosis (double positive cells) in splenic lymphocytes treated with kainic acid alone. However, melatonin treated in combination with kainic acid, showed attenuation to apoptosis and necrosis on splenic lymphocytes. This study depicts that kainic acid induced inflammatory toxicity could be attenuated by exogenous melatonin treatment as evident by the decreased levels of the inflammatory cytokines, immune reactions and free radical production.

Increased Seizure Susceptibility in a Mouse with Diacylglycerol Kinase <i>β</i> Deficiency

Diacylglycerol kinase (DGK) is an enzyme that converts diacylglycerol to phosphatidic acid. Several DGK isoforms have been implicated in the pathogenesis of seizure, but the role of DGKβ in seizure is unknown. In the present study, we investigated the involvement of DGKβ in seizure using DGKβ knockout (KO) mice. Seizures were more severe in DGKβ KO mice than in wild-type (WT) mice after pentylenetetrazol (PTZ) treatment and after kainic acid treatment, but there were no differences in latency to seizure. The expression levels of DGKβ in the hippocampal CA1, CA3, or DG areas did not differ between PTZ (60 mg/kg) treatment and saline treatment. There were fewer parvalbumin-positive interneurons in the hippocampal CA3 area in DGKβ KO mice than in control WT mice, which might partly account for the increased seizure susceptibility displayed by DGKβ KO mice. These results suggest that DGKβ may play a pivotal role in the development of the relevant interneurons, and that on inherent deficiency of DGKβ increases the animal’s sensitivity to seizure-inducing stimuli.

Characterization of the expression of macrophage inflammatory protein‐1α (MIP‐1α) and C‐C chemokine receptor 5 (CCR5) after kainic acid‐induced status epilepticus (SE) in juvenile rats

To identify the potential role of macrophage inflammatory protein-1α (MIP-1α) with its C-C chemokine receptor 5 (CCR5) in epileptogenic brain injury, we examined their expression in juvenile rat hippocampus and explored the potential link between MIP-1α, CCR5 and neuropathological alterations after status epilepticus (SE) induced by intracerebroventricular (i.c.v.) kainic acid (KA) injection. Based on the determination of the development of spontaneous seizures initiated by SE in developing rat brain, we firstly examined hippocampal neurone damage through Nissl and Fluoro-Jade B staining, and evaluated microglial reaction during the early phase following KA-induced SE in 21-day-old rats. MIP-1α and CCR5 protein were quantified by ELISA and Western blot respectively following mRNA by real-time PCR. We also mapped MIP-1α and CCR5 expression in the hippocampus by immunohistochemistry and identified their cellular sources using double-labelling immunofluorescence. In juvenile rats, KA caused characteristic neurone damage in the hippocampal subfields, with accompanying microglial accumulation. In parallel with mRNA expression, MIP-1α protein in hippocampus was transiently increased after KA treatment, and peaked from 16 to 72 h. Double-labelling immunofluorescence revealed that MIP-1α was localized to microglia. Up-regulated CCR5 remained prominent at 24 and 72 h and was mainly localized to activated microglia. Further immunohistochemistry revealed that MIP-1α and CCR5 expression were closely consistent with microglial accumulation in corresponding hippocampal subfields undergoing degenerative changes. Our data indicated that MIP-1α as a regulator, linking with the CCR5 receptor, may be involved within the early stages of the epileptogenic process following SE by i.c.v. KA injection.

Expression of CD200 in alternative activation of microglia following an excitotoxic lesion in the mouse hippocampus

CD200 is a glycoprotein that is expressed on the surfaces of neurons and other cells. It interacts with its receptor, CD200R, which is expressed on cells of the myeloid lineage, including microglia. The interaction of CD200 with its receptor plays a significant role in maintaining microglia in a quiescent state; thus, a decrease in CD200 expression in the brain is associated with evidence of microglial activation. However, their roles in pathological progression remain unclear. We examined the expression of CD200 in kainic acid (KA)-induced neurodegeneration of the mouse hippocampus. Our quantitative analysis revealed that CD200 was constitutively expressed in the normal brain and transiently upregulated by KA treatment. At the cellular level, CD200 was expressed in neurons in control, and was upregulated primarily in the microglia of KA-treated mouse hippocampi. We examined the contribution of CD200 to both the classical and alternative activation of microglia in vitro using an adult microglia culture, which was exposed to interleukin-4 (IL-4) with and without lipopolysaccharide (LPS). CD200 expression was increased after exposure to IL-4, but not to LPS. These in vivo experiments demonstrated that CD200 was transiently expressed in microglia in a process mediated by the inflammatory response. Based on CD200R expression in microglia, it suggests that microglia is maintained in an activated state with autocrine signaling by interactions between microglial CD200 and its CD200R. Moreover, we suggest that CD200 may be expressed in the alternative activation of microglia and play a beneficial role in neuroinflammation.

Neuroprotection from Tissue Inhibitor of Metalloproteinase-1 and its nanoparticles

Matrix metalloproteinases (MMPs) are family of zinc dependent endopeptidases, which cleave extracellular matrix proteins, and play an important role in tissue remodelling in physiological and pathological processes. There is enhanced expression of MMPs, in particular MMP-9, during numerous pathological conditions, including epilepsy and ischemic stroke. Therefore, inhibition of MMP-9 is considered as a potential therapeutic target. Tissue Inhibitor of Matrix Metalloproteinase-1 (TIMP-1) is a 28kDa endogenous inhibitor of MMP-9. In this study we examined recombinant mouse TIMP-1 for its in-vitro neuroprotective effects, against Kainic Acid (KA) induced excitotoxicity in organotypic hippocampal slice culture (OHC) model. We also studied, sustained release effects of TIMP-1 in OHC by using poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs). TIMP-1 and TIMP-1 PLGA NPs were added to the slice cultures at different time points, i.e., 30min before treatment with KA and 6h after KA treatment. Propidium iodide staining was used to reveal cell toxicity in the cultures. In addition, neurotoxicity was assessed using standard lactate dehydrogenase (LDH) release assay. Gelatinolytic activity in conditioned cultured medium of OHC was accessed by a fluorescent substrate assay. Briefly, our result show that TIMP-1 provided significant level of neuroprotection, especially when given before 30min of KA and released from the NPs. Since gelatinolytic activity assay showed a decrease in MMP-9 activity, it can be suggested that this neuroprotection might be mediated by the gelatinase inhibition.

P53 Mediates autophagy activation and mitochondria dysfunction in kainic acid-induced excitotoxicity in primary striatal neurons

The present study sought to investigate if p53 mediates autophagy activation and mitochondria dysfunction in primary striatal neurons in kainic acid (KA)-induced excitotoxicity. The excitotoxic model of primary striatal neurons was established with KA. The levels of p53, microtubule-associated protein 1 light chain 3 (LC3), Beclin1, and p62 were examined by Western blot and immunostaining. Autophagy activation was also determined with electron microscope. To evaluate the contribution of p53 to autophagy activation and mitochondria dysfunction in KA-induced excitotoxicity, the protein levels of LC3, Beclin1, and p62, the mitochondrial transmembrane potential and the mitochondrial Reactive oxygen species (ROS) after pretreatment with the p53 inhibitor pifithrin-alpha (PFT-α) and the autophagy inhibitor 3-methyladenine (3-MA) were analyzed. Excitotoxic neuronal injury was induced after KA treatment as demonstrated by increases in lactate dehydrogenase (LDH) leakage and was significantly inhibited by PFT-α. Western blot and immunostaining showed that the induction of p53 protein occurred in the cytosol and the nucleus. Increases in autophagic proteins LC3 and Beclin1 were observed, whereas the protein levels of p62 decreased after KA treatment. Electron microscope analysis showed increased autophagosomes in the cytoplasm. The changes in LC3, Beclin1, and p62 levels were blocked by PFT-α, PFT-μ, 3-MA, and E64d but not Z-DEVD-FMK. JC-1 staining showed the depolarization of mitochondrial membrane potential after excitotoxic insult. Mito-tracker and RedoxSensor Red CC-1 staining showed an increased production of mitochondrial ROS after excitotoxic insult. These effects were significantly suppressed after pretreatment with PFT-α and 3-MA. This study suggests that p53 mediates KA-induced autophagy activation and mitochondrial dysfunction in striatal neurons.

Matrix metalloproteinase-7 regulates cleavage of pro-nerve growth factor and is neuroprotective following kainic acid-induced seizures.

The neurotrophin nerve growth factor (NGF) regulates neuronal growth, differentiation, and survival during development. However, the precursor of NGF, proNGF, is a potent apoptotic ligand for the p75 neurotrophin receptor (p75(NTR))-sortilin complex. The mechanisms that regulate cleavage of proNGF, therefore, are critical determinants of whether this factor promotes neuronal survival or death. In this study, we demonstrate that, following kainic acid-induced seizures, the proNGF processing enzyme matrix metalloproteinase 7 (MMP-7) and its inhibitor TIMP-1 (tissue inhibitor of matrix metalloproteinase 1) are regulated in a manner that prevents proneurotrophin cleavage and leads to increased proNGF in the extracellular milieu. Furthermore, we demonstrate both in vitro and in vivo that exogenous MMP-7 enhances proNGF cleavage and provides neuroprotection following kainic acid treatment. These data demonstrate that increased extracellular proNGF levels following seizures are stabilized by altered MMP-7 enzymatic activity, leading to increased neuronal death via activation of p75(NTR).

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.

Involvement of cyclin-dependent kinase-5 in the kainic acid-mediated degeneration of glutamatergic synapses in the rat hippocampus

Increased levels of glutamate causing excitotoxic damage accompany neurological disorders such as ischemia/stroke, epilepsy and some neurodegenerative diseases. Cyclin-dependent kinase-5 (Cdk5) is important for synaptic plasticity and is deregulated in neurodegenerative diseases. However, the mechanisms by which kainic acid (KA)-induced excitotoxic damage involves Cdk5 in neuronal injury are not fully understood. In this work, we have thus studied involvement of Cdk5 in the KA-mediated degeneration of glutamatergic synapses in the rat hippocampus. KA induced degeneration of mossy fiber synapses and decreased glutamate receptor (GluR)6/7 and post-synaptic density protein 95 (PSD95) levels in rat hippocampus in vivo after intraventricular injection of KA. KA also increased the cleavage of Cdk5 regulatory protein p35, and Cdk5 phosphorylation in the hippocampus at 12 h after treatment. Studies with hippocampal neurons in vitro showed a rapid decline in GluR6/7 and PSD95 levels after KA treatment with the breakdown of p35 protein and phosphorylation of Cdk5. These changes depended on an increase in calcium as shown by the chelators 1,2-bis(o-aminophenoxy)ethane-N,N,N ',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and glycol-bis (2-aminoethylether)-N,N,N ',N '-tetra-acetic acid. Inhibition of Cdk5 using roscovitine or employing dominant-negative Cdk5 and Cdk5 silencing RNA constructs counteracted the decreases in GluR6/7 and PSD95 levels induced by KA in hippocampal neurons. The dominant-negative Cdk5 was also able to decrease neuronal degeneration induced by KA in cultured neurons. The results show that Cdk5 is essentially involved in the KA-mediated alterations in synaptic proteins and in cell degeneration in hippocampal neurons after an excitotoxic injury. Inhibition of pathways activated by Cdk5 may be beneficial for treatment of synaptic degeneration and excitotoxicity observed in various brain diseases.

Oral Uncaria rhynchophylla (UR) reduces kainic acid-induced epileptic seizures and neuronal death accompanied by attenuating glial cell proliferation and S100B proteins in rats

Epilepsy is a common clinical syndrome with recurrent neuronal discharges in cerebral cortex and hippocampus. Here we aim to determine the protective role of Uncaria rhynchophylla (UR), an herbal drug belong to Traditional Chinese Medicine (TCM), on epileptic rats. To address this issue, we tested the effect of UR on kainic acid (KA)-induced epileptic seizures and further investigate the underlying mechanisms. Oral UR successfully decreased neuronal death and discharges in hippocampal CA1 pyramidal neurons. The population spikes (PSs) were decreased from 4.1 ± 0.4 mV to 2.1 ± 0.3 mV in KA-induced epileptic seizures and UR-treated groups, respectively. Oral UR protected animals from neuronal death induced by KA treatment (from 34 ± 4.6 to 191.7 ± 48.6 neurons/field) through attenuating glial cell proliferation and S100B protein expression but not GABAA and TRPV1 receptors. The above results provide detail mechanisms underlying the neuroprotective action of UR on KA-induced epileptic seizure in hippocampal CA1 neurons.