Systemic Injection Of Kainic Acid Research Articles

TMT-based proteomics profile reveals changes of the entorhinal cortex in a kainic acid model of epilepsy in mice

Experimental modeling and clinical neuroimaging of patients has shown that certain seizures are capable of causing neuronal death. Research into cell death after seizures has identified the induction of the molecular machinery of apoptosis. Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults, which is characterized by substantial pathological abnormalities in the temporal lobe, including the hippocampus and entorhinal cortex (EC). Although decades of studies have revealed numerous molecular abnormalities in the hippocampus that are linked to TLE, the biochemical mechanisms associated with TLE in EC remain unclear.In this study, we explored these early phenotypical alterations in the EC 5 days after mice were given a systemic injection of kainic acid (KA) to induce status epilepticus (KA-SE). we used the Tandem Mass Tag (TMT) combined with LC-MS/MS approach to identify distinct proteins in the EC in a mouse model of KA-SE model.According to the findings, 355 differentially abundant proteins including 199 upregulated and 156 downregulated differentially abundant proteins were discovered. The first-ranked biological process according to Gene Ontology (GO) analysis was “negative control of extrinsic apoptotic signaling”. “Apoptosis” was the most significantly enriched Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway. Compared with those in control mice, BCL2L1, NTRK2 and MAPK10 abundance levels were reduced in KA mice. MAPK10 and NTRK2 act as upstream regulators to regulate BCL2L1, and BCL2L1 Inhibits cell death by blocking the voltage- dependent anion channel (VDAC) and preventing the release of the caspase activator, CYC1, from the mitochondrial membrane. However, ITPR1 was increased at the mRNA and protein levels in KA mice. Furthermore, there was no significant difference in ACTB, TUBA1A and TUBA4A levels between the two groups. Our results offer clues to help identify biomarkers for the development of pharmacological therapies targeted at epilepsy.

A role for astrocyte-derived amyloid β peptides in the degeneration of neurons in an animal model of temporal lobe epilepsy.

Kainic acid, an analogue of the excitatory neurotransmitter glutamate, can trigger seizures and neurotoxicity in the hippocampus and other limbic structures in a manner that mirrors the neuropathology of human temporal lobe epilepsy (TLE). However, the underlying mechanisms associated with the neurotoxicity remain unclear. Since amyloid-β (Aβ) peptides, which are critical in the development of Alzheimer's disease, can mediate toxicity by activating glutamatergic NMDA receptors, it is likely that the enhanced glutamatergic transmission that renders hippocampal neurons vulnerable to kainic acid treatment may involve Aβ peptides. Thus, we seek to establish what role Aβ plays in kainic acid-induced toxicity using in vivo and in vitro paradigms. Our results show that systemic injection of kainic acid to adult rats triggers seizures, gliosis and loss of hippocampal neurons, along with increased levels/processing of amyloid precursor protein (APP), resulting in the enhanced production of Aβ-related peptides. The changes in APP levels/processing were evident primarily in activated astrocytes, implying a role for astrocytic Aβ in kainic acid-induced toxicity. Accordingly, we showed that treating rat primary cultured astrocytes with kainic acid can lead to increased Aβ production/secretion without any compromise in cell viability. Additionally, we revealed that kainic acid reduces neuronal viability more in neuronal/astrocyte co-cultures than in pure neuronal culture, and this is attenuated by precluding Aβ production. Collectively, these results indicate that increased production/secretion of Aβ-related peptides from activated astrocytes can contribute to neurotoxicity in kainic acid-treated rats. Since kainic acid administration can lead to neuropathological changes resembling TLE, it is likely that APP/Aβ peptides derived from astrocytes may have a role in TLE pathogenesis.

Targeting brain and peripheral plasticity of the lipidome in acute kainic acid-induced epileptic seizures in mice via quantitative mass spectrometry

Epilepsy is a highly common chronic neurological disorder, manifested in many different types, affecting ~1% of the worldwide human population. The molecular mechanisms of epileptogenesis have not yet been clarified, and pharmacoresistance exhibited by 30–40% of epilepsy patients remains a major obstacle in medical care. Growing evidence indicates a role of lipid signalling pathways in epileptogenesis, thus lipid signals emerge as potential biomarkers for the onset and evolving course of the epileptic disorder, as well as potential therapeutic agents and targets. For this purpose, we applied a lipidomic strategy to unravel lipid alterations in brain regions, periphery tissues and plasma that are specific for acute epileptic seizures in mice at 1h after seizure induction by systemic kainic acid injection as compared to vehicle controls. Specifically, levels of (i) selected phospholipids and sphingomyelins, (ii) the endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), and the endocannabinoid-related compounds oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), (iii) arachidonic acid (AA), (iv) selected eicosanoids, and (v) fatty acyl content of lipidome were determined in pulverized tissues from six brain regions of kainic acid induced epileptic seizure models and vehicle controls: hypothalamus, hippocampus, thalamus, striatum, cerebellum and cerebral cortex, and from peripheral organs, such as heart and lungs, and in plasma. Alterations in lipid levels after acute epileptic seizures as compared to non-seizure controls were found to be brain region- and periphery tissue-specific, including specific plasma lipid correlates, highlighting their value as marker candidates in translational research studies, and/or drug discovery and response monitoring.

CA3 Synaptic Silencing Attenuates Kainic Acid-Induced Seizures and Hippocampal Network Oscillations.

Epilepsy is a neurological disorder defined by the presence of seizure activity, manifest both behaviorally and as abnormal activity in neuronal networks. An established model to study the disorder in rodents is the systemic injection of kainic acid, an excitatory neurotoxin that at low doses quickly induces behavioral and electrophysiological seizures. Although the CA3 region of the hippocampus has been suggested to be crucial for kainic acid-induced seizure, because of its strong expression of kainate glutamate receptors and its high degree of recurrent connectivity, the precise role of excitatory transmission in CA3 in the generation of seizure and the accompanying increase in neuronal oscillations remains largely untested. Here we use transgenic mice in which CA3 pyramidal cell synaptic transmission can be inducibly silenced in the adult to demonstrate CA3 excitatory output is required for both the generation of epileptiform oscillatory activity and the progression of behavioral seizures.

Quinazoline-based tricyclic compounds that regulate programmed cell death, induce neuronal differentiation, and are curative in animal models for excitotoxicity and hereditary brain disease.

Expanding on a quinazoline scaffold, we developed tricyclic compounds with biological activity. These compounds bind to the 18 kDa translocator protein (TSPO) and protect U118MG (glioblastoma cell line of glial origin) cells from glutamate-induced cell death. Fascinating, they can induce neuronal differentiation of PC12 cells (cell line of pheochromocytoma origin with neuronal characteristics) known to display neuronal characteristics, including outgrowth of neurites, tubulin expression, and NeuN (antigen known as ‘neuronal nuclei’, also known as Rbfox3) expression. As part of the neurodifferentiation process, they can amplify cell death induced by glutamate. Interestingly, the compound 2-phenylquinazolin-4-yl dimethylcarbamate (MGV-1) can induce expansive neurite sprouting on its own and also in synergy with nerve growth factor and with glutamate. Glycine is not required, indicating that N-methyl-D-aspartate receptors are not involved in this activity. These diverse effects on cells of glial origin and on cells with neuronal characteristics induced in culture by this one compound, MGV-1, as reported in this article, mimic the diverse events that take place during embryonic development of the brain (maintenance of glial integrity, differentiation of progenitor cells to mature neurons, and weeding out of non-differentiating progenitor cells). Such mechanisms are also important for protective, curative, and restorative processes that occur during and after brain injury and brain disease. Indeed, we found in a rat model of systemic kainic acid injection that MGV-1 can prevent seizures, counteract the process of ongoing brain damage, including edema, and restore behavior defects to normal patterns. Furthermore, in the R6-2 (transgenic mouse model for Huntington disease; Strain name: B6CBA-Tg(HDexon1)62Gpb/3J) transgenic mouse model for Huntington disease, derivatives of MGV-1 can increase lifespan by >20% and reduce incidence of abnormal movements. Also in vitro, these derivatives were more effective than MGV-1.

Kainic acid induces expression of caveolin-1 in activated microglia in rat brain

Caveolin-1, a major constituent of caveolae, has been implicated in endocytosis, signal transduction and cholesterol transport in a wide variety of cells. In the present study, the expression of caveolin-1 was examined by immunohistochemistry in rat brain with or without systemic injection of kainic acid (KA). Caveolin-1 immunoreactivity was observed in capillary walls in brains of control rats. From one to seven days after KA injection, caveolin-1 immunoreactivity appeared in activated microglia in the cerebral cortex, hippocampus and other brain regions. The strongest immunoreactivity of microglia was seen after 3 days after KA administration. The expression of caveolin-1 was confirmed by RT-PCR and Western blot analysis, respectively. The induction of caveolin-1 expression in microglia activated in response to kainic acid administration suggests its possible role in a modulation of inflammation.

Systemic Injection of Kainic Acid Differently Affects LTP Magnitude Depending on its Epileptogenic Efficiency

Seizures have profound impact on synaptic function and plasticity. While kainic acid is a popular method to induce seizures and to potentially affect synaptic plasticity, it can also produce physiological-like oscillations and trigger some forms of long-term potentiation (LTP). Here, we examine whether induction of LTP is altered in hippocampal slices prepared from rats with different sensitivity to develop status epilepticus (SE) by systemic injection of kainic acid. Rats were treated with multiple low doses of kainic acid (5 mg/kg; i.p.) to develop SE in a majority of animals (72–85% rats). A group of rats were resistant to develop SE (15–28%) after several accumulated doses. Animals were subsequently tested using chronic recordings and object recognition tasks before brain slices were prepared for histological studies and to examine basic features of hippocampal synaptic function and plasticity, including input/output curves, paired-pulse facilitation and theta-burst induced LTP. Consistent with previous reports in kindling and pilocapine models, LTP was reduced in rats that developed SE after kainic acid injection. These animals exhibited signs of hippocampal sclerosis and developed spontaneous seizures. In contrast, resistant rats did not become epileptic and had no signs of cell loss and mossy fiber sprouting. In slices from resistant rats, theta-burst stimulation induced LTP of higher magnitude when compared with control and epileptic rats. Variations on LTP magnitude correlate with animals’ performance in a hippocampal-dependent spatial memory task. Our results suggest dissociable long-term effects of treatment with kainic acid on synaptic function and plasticity depending on its epileptogenic efficiency.

Cellular Source of Apolipoprotein E4 Determines Neuronal Susceptibility to Excitotoxic Injury in Transgenic Mice

The lipid transport protein apolipoprotein E (apoE) is abundantly expressed in the brain. Its main isoforms in humans are apoE2, apoE3, and apoE4. ApoE4 is the major known genetic risk factor for Alzheimer's disease and also contributes to the pathogenesis of various other neurological conditions. In the central nervous system, apoE is synthesized by glial cells and neurons, but it is unclear whether the cellular source affects its biological activities. To address this issue, we induced excitotoxic injury by systemic kainic acid injection in transgenic Apoe knockout mice expressing human apoE isoforms in astrocytes or neurons. Regardless of its cellular source, apoE3 expression protected neuronal synapses and dendrites against the excitotoxicity seen in apoE-deficient mice. Astrocyte-derived apoE4, which has previously been shown to have detrimental effects in vitro, was as excitoprotective as apoE3 in vivo. In contrast, neuronal expression of apoE4 was not protective and resulted in loss of cortical neurons after excitotoxic challenge, indicating that neuronal apoE4 promotes excitotoxic cell death. Thus, an imbalance between astrocytic (excitoprotective) and neuronal (neurotoxic) apoE4 expression may increase susceptibility to diverse neurological diseases involving excitotoxic mechanisms.

C‐Jun N‐terminal kinase signaling pathway in excitotoxic cell death following kainic acid‐induced status epilepticus

Systemic injections of kainic acid (KA) cause epileptic seizures with delayed neuronal damage in the limbic system, particularly in the hippocampus. KA excitotoxicity activates complex signal transduction events that trigger apoptotic cell death. The c-Jun N-terminal kinase (JNK) pathway plays an important role in cell death, and the peptide D-JNKI1, a competitive JNK inhibitor, is a potent neuroprotective agent. To analyse the role of JNK and the effects of D-JNKI1 administration on excitotoxic neuronal death, we induced epileptic seizures by intraperitoneal (i.p.) injection of KA in adult male Sprague-Dawley rats; a group of rats received i.p. D-JNKI1 2 h after KA. KA caused massive cell death in the hippocampus: in Nissl-stained sections, stereological counts showed a significant decrease in neuronal density in all CA fields, both at 1 and 5 days after seizures, which was partially prevented by D-JNKI1 treatment. These results were confirmed by counts of degenerating neurons in CA3 in FluoroJade B-stained sections. Seizure activity also induced marked gliosis as observed with glial fibrillary acidic protein (GFAP) immunohistochemistry. We also analysed c-Jun activation as a target of JNK and central transcriptional effector in the adult rat brain following KA injection. Phospho-c-Jun immunoreactivity was absent in the hippocampus of untreated animals, whereas strong nuclear neuronal labeling could be observed, starting from 3 h after KA administration, in microtubule-associated protein-2-positive neurons but not in GFAP-positive astrocytes. D-JNKI1 treatment also reduced the positivity for phospho-c-Jun in the hippocampus, thus confirming the specificity of the peptide in blocking JNK. Therefore, JNK is a promising target for blocking seizure-induced cell death.

Effect of age on kainate-induced seizure severity and cell death

While the onset and extent of epilepsy increases in the aged population, the reasons for this increased incidence remain unexplored. The present study used two inbred strains of mice (C57BL/6J and FVB/NJ) to address the genetic control of age-dependent neurodegeneration by building upon previous experiments that have identified phenotypic differences in susceptibility to hippocampal seizure-induced cell death. We determined if seizure induction and seizure-induced cell death are affected differentially in young adult, mature, and aged male C57BL/6J and FVB/NJ mice administered the excitotoxin, kainic acid. Dose response testing was performed in three to four groups of male mice from each strain. Following kainate injections, mice were scored for seizure activity and brains from mice in each age group were processed for light microscopic histopathologic evaluation 7 days following kainate administration to evaluate the severity of seizure-induced brain damage. Irrespective of the dose of kainate administered or the age group examined, resistant strains of mice (C57BL/6J) continued to be resistant to seizure-induced cell death. In contrast, aged animals of the FVB/NJ strain were more vulnerable to the induction of behavioral seizures and associated neuropathology after systemic injection of kainic acid than young or middle-aged mice. Results from these studies suggest that the age-related increased susceptibility to the neurotoxic effects of seizure induction and seizure-induced injury is regulated in a strain-dependent manner, similar to previous observations in young adult mice.

Involvement of endogenous prostaglandin F 2α on kainic acid-induced seizure activity through FP receptor: The mechanism of proconvulsant effects of COX-2 inhibitors

COX-2 and prostaglandins (PGs) might play important roles in epilepsy. In kainic acid-induced seizures, the brain largely increases PGD 2, first from COX-1 and later COX-2-induced PGF 2α. Pre-treatment with COX-2 inhibitors such as indomethacin, nimesulide, and celecoxib is known to aggravate kainic acid (KA)-induced seizure activity. However it is not known whether the proconvulsant effect of those non-steroidal anti-inflammatory drugs (NSAIDs) is due to changes in endogenous prostaglandins (PGs), or what types of PGs are involved. The purpose of this study was to determine the effect of intracisternally administered PGs on KA-induced seizures aggravated by pre- or post-treatment with COX-2 inhibitors. Systemic KA injection (10 mg/kg i.p.) in mice evoked mild seizure activity within 15 min. PGs were administrated intracisternally 20 min prior to KA administration. COX inhibitors (indomethacin, nimesulide, and ketoprofen, 10 mg/kg i.p.) were injected 1 h before or 15 min after KA. An additional COX-2 inhibitor, celecoxib, was administered orally. Intracisternally administered PGF 2α (700 ng), but not PGD 2 (700 ng) or PGE 2 (700 ng) completely alleviated KA-induced seizures potentiated by COX-2 inhibitors, and also reduced KA-induced hippocampal neuronal death aggravated by indomethacin. PGF 2α alone did not affect KA-induced seizures. However, an FP receptor antagonist, AL 8810 (10 or 50 ng) which is an 11β-fluoro analogue of PGF 2α potentiated KA-induced seizure activity dose-dependently. In summary, pre- or post-treatment with COX-2 inhibitors aggravates KA-induced seizures, which suggests to change the endogenous PGF 2α. Seizure-induced PGF 2α might act as an endogenous anticonvulsant through FP receptors.

Microarray profile of seizure damage-refractory hippocampal CA3 in a mouse model of epileptic preconditioning

A neuroprotected state can be acquired by preconditioning brain with a stimulus that is subthreshold for damage (tolerance). Acquisition of tolerance involves coordinate, bi-directional changes to gene expression levels and the re-programmed phenotype is determined by the preconditioning stimulus. While best studied in ischemic brain there is evidence brief seizures can confer tolerance against prolonged seizures (status epilepticus). Presently, we developed a model of epileptic preconditioning in mice and used microarrays to gain insight into the transcriptional phenotype within the target hippocampus at the time tolerance had been acquired. Epileptic tolerance was induced by an episode of non-damaging seizures in adult C57Bl/6 mice using a systemic injection of kainic acid. Neuron and DNA damage-positive cell counts 24 h after status epilepticus induced by intraamygdala microinjection of kainic acid revealed preconditioning given 24 h prior reduced CA3 neuronal death by ∼45% compared with non-tolerant seizure mice. Microarray analysis of over 39,000 transcripts (Affymetrix 430 2.0 chip) from microdissected CA3 subfields was undertaken at the point at which tolerance was acquired. Results revealed a unique profile of small numbers of equivalently up- and down-regulated genes with biological functions that included transport and localization, ubiquitin metabolism, apoptosis and cell cycle control. Select microarray findings were validated post hoc by real-time polymerase chain reaction and Western blotting. The present study defines a paradigm for inducing epileptic preconditioning in mice and first insight into the global transcriptome of the seizure-damage refractory brain.

Effects of Manganese Complexes of Curcumin and Diacetylcurcumin on Kainic Acid-Induced Neurotoxic Responses in the Rat Hippocampus

This study aimed to investigate the mechanism underlying the protective effects of manganese complexes of curcumin (Cp-Mn) and diacetylcurcumin (DiAc-Cp-Mn) on kainic acid (KA)-induced excitotoxicity in the rat hippocampus. Systemic injection of KA (10 mg/kg, i.p.) caused seizures and increased the expression of neurotoxic markers, immediate early genes [c-jun, cyclooxygenase 2 (COX-2), brain-derived neurotrophic factor (BDNF), and heat shock protein 70 (hsp70)] and a delayed response gene [inducible nitric oxide synthase (iNOS)], which were measured at 6 and 72 h after KA injection, respectively, in the hippocampus. Pretreatment with Cp-Mn (50 mg/kg, i.p.) and DiAc-Cp-Mn (50 mg/kg, i.p.) but not with curcumin (50 mg/kg, i.p.) delayed the onset of KA-induced seizure without affecting the seizure score. KA injection induced c-Fos immunoreactivity in DG, CA1, and CA3 hippocampal regions, the expression of which peaked at 6 h after injection. Cp-Mn and DiAc-Cp-Mn treatment significantly decreased c-Fos expression elicited by KA. Moreover, Cp-Mn and DiAc-Cp-Mn administration suppressed the KA-induced expression of c-jun, COX-2, BDNF, and iNOS mRNA, whereas curcumin attenuated only iNOS mRNA expression. No compounds tested had an effect on KA-induced hsp70 expression. It is therefore likely that in addition to radical scavenging and SOD-like activities, the suppression of potential neuronal injury marker expression by Cp-Mn and DiAc-Cp-Mn, contributes to the neuroprotective activities of these compounds, which are superior to those of curcumin, on KA-induced excitotoxicity in the hippocampus. These results suggest the beneficial effects of Cp-Mn, and DiAc-Cp-Mn on the treatment of excitotoxicity-induced neurodegenerative diseases.

Hippocalcin protects hippocampal neurons against excitotoxin damage by enhancing calcium extrusion

Hippocalcin, which is a member of the neuronal calcium-sensor protein family, is highly expressed in hippocampal pyramidal cells. Recently, it was demonstrated that hippocalcin deficit caused an increase in neuronal cell death in the field CA3 of Ammon’s horn (CA3) region of the hippocampus following the systemic injection of kainic acid. Treatment with kainic acid results in seizure-induced cell death in CA3. In the present study, we injected quinolinic acid, which is an N-methyl- d-aspartate receptor agonist, into the hippocampal field CA1 of Ammon’s horn (CA1) region in hippocalcin-knockout (−/−) mice, a procedure which mimics transient ischemia. Although significant pyknotic changes were observed at the injected site in wild-type (+/+) mice 24 h after injection, the area of pyknotic cells extended throughout the hippocampus in −/− mice. The quantification of cell numbers in Nissl-stained sections indicated that the cell damage in −/− mice was more severe than that in +/+ mice. The density of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick-end labeling-positive cells roughly paralleled that of Nissl-stained pyknotic cells. Primary cultures of hippocampal neurons showed that the number of surviving neurons from −/− mice after 7 days in culture was smaller than the number from +/+ mice. The measurement of intracellular calcium concentrations in single cells revealed that the calcium extrusion from −/− neurons was slower than that from +/+ neurons. The involvement of hippocalcin in the upkeep of calcium extrusion was confirmed using hippocalcin-expressing COS7 cells. These results suggest that hippocalcin plays an important role in calcium extrusion from neurons and, in turn, helps to protect them against calcium-dependent excitotoxin damage in the hippocampus.

Occurrence of complement protein C3 in dying pyramidal neurons in rat hippocampus after systemic administration of kainic acid

To evaluate the roles of complement in kainic acid (KA)-induced neuronal damages, the immunohistochemical localization of the complement protein C3 was examined in rat hippocampus after systemic KA injection. The immunoreactivity for C3 was found in glial cells in control rats, and such glial cells were increased in number after KA injection. Our confocal study showed that C3-positive glial cells were microglia. Three to seven days after KA, C3 immunoreactivity appeared in CA1 and CA3 pyramidal neurons. Double staining for C3 combined with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling showed that occurrence of C3 immunoreactivity in neurons coincided well with that of DNA fragmentation. Western blot analysis and RT-PCR experiments suggested local synthesis of C3 by brain cells. Our results suggest that C3 contributes greatly to neuronal death after systemic KA administration, and that microglia and neurons are the local source of C3 in KA-induced brain injury.

A mechanism for the inactivation of Ca 2+/calmodulin-dependent protein kinase II during prolonged seizure activity and its consequence after the recovery from seizure activity in rats in vivo

Seizure is a form of excessive neuronal excitation and seizure-induced neuronal damage has profound effects on the prognosis of epilepsy. In various seizure models, the inactivation of Ca 2+/calmodulin-dependent protein kinase II (CaMKII) occurs during seizure activity preceding neuronal cell death. CaMKII is a multifunctional protein kinase enriched in the brain and involved in various ways the regulation of neuronal activity. CaMKII inactivation during seizure activity may modify neuronal cell survival after seizure. However, the mechanism for CaMKII inactivation and its consequence after seizure recovery remain to be elucidated yet. In the present study, we employed a prolonged seizure model by systemic injection of kainic acid into rats and biochemically examined the activity state of CaMKII. In status epilepticus induced by kainic acid, not only the inactivation of CaMKII in brain homogenate, but also a shift in the distribution of CaMKII protein from the soluble to particulate fraction occurred in both hippocampus and parietal cortex. The particulate CaMKII showed a large decrease in the specific activity and a concurrent large increase in the autophosphorylation ratio at Thr-286 (α) and at Thr-287 (β). In contrast, the soluble CaMKII showed normal or rather decreased specific activity and autophosphorylation ratio. After 24 h of recovery from kainic acid-induced status epilepticus, all such changes had disappeared. On the other hand, the total amount of CaMKII was decreased by 35% in hippocampus and 20% in parietal cortex, but the existing CaMKII was indistinguishable from those of controls in terms of the autonomous activity ratio, specific activity and autophosphorylation ratio. Thus, CaMKII inactivation in kainic acid-induced status epilepticus seems to be derived not from simple degradation of the enzyme, but from the formation of the autophosphorylated, inactivated and sedimentable CaMKII. Such a form of CaMKII may be important during pathological conditions in vivo in preventing excessive CaMKII activation due to Ca 2+ overload.

Inflammatory Response and Glia Activation in Developing Rat Hippocampus after Status Epilepticus

We investigated the activation of microglia and astrocytes, induction of cytokines, and hippocampal neuronal damage, 4 and 24 h after kainic acid-induced status epilepticus (SE) in postnatal day (PN) 9, 15, and 21 rats. Limbic seizures were induced by systemic injection of kainic acid. Glia activation and neuronal cell loss were studied by using immunocytochemistry and Western blot. Cytokine expression was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by Southern blot quantification. After SE onset, hippocampal glia activation, cytokine expression, and neuronal damage are all age-dependent phenomena. In the hippocampus, neuronal injury occurs only when cytokines are induced in glia, and cytokine synthesis precedes the appearance of degenerating neurons. Neuronal injury is more pronounced when interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are produced in addition to IL-1beta. This study shows that cytokine induction in rat brain after sustained seizures is age dependent, and it is associated with the appearance of cell injury.

Up-regulation of peripherin is associated with alterations in synaptic plasticity in CA1 and CA3 regions of hippocampus

Peripherin is a type III intermediate filament protein normally undetectable in most brain neurons. Here, we report a similar pattern of peripherin expression in the brains of both mice treated with systemic injections of kainic acid (KA) and in peripherin transgenic mice (Per mice) over-expressing the normal peripherin gene under its own promoter. Double-immunofluorescence labeling revealed a partial co-localization of peripherin with the microtubule-associated protein MAP2, but not with neurofilament proteins. Electrophysiological studies revealed that synaptic plasticity was markedly altered in Per mice: in CA1, long-term potentiation (LTP) was decreased in Per slices (+29 +/- 2.0%, vs. +58 +/- 5.4%, in WT); while in CA3, LTP was increased in Per (+63 +/- 3.5% vs. +43 +/- 2.4.0%). In the hippocampus of Per mice, the levels of MAP2 were decreased, though synaptophysin and PSD95 remained unchanged. These intriguing findings suggest a role of peripherin in the alteration of hippocampal synaptic plasticity.

Response to kainic acid injections: changes in staining for zinc, FOS, cell death and glial response in the rat forebrain

A pool of zinc is present in synaptic vesicles in a population of glutamatergic neurones. Zinc appears to modulate synaptic transmission and cause neuronal death. The status of vesicular zinc, neuronal death and glial reaction in the rat forebrain was analysed after a systemic injection of kainic acid in order to establish a model for future studies on zinc function. Rats received a systemic injection of kainic acid (10 mg/kg) and were killed 3, 6, 12, 24 and 48 h post-treatment. Timm's method and zinquin staining were used to detect zinc. Immunostaining for Fos-like proteins and staining with Fluoro-Jade B were used to detect cell reaction and degeneration, respectively. Glial fibrillary acidic protein and tomato lectin were used as glial markers. Zinquin staining for zinc rose transitorily in neuronal somata 6 h after injection (not observed at 24–48 h) in the piriform and entorhinal cortices, amygdala and hippocampus. In contrast sulphide/silver staining for zinc showed virtually no rise in cytoplasmic zinc (except in cornus ammonis field 1 of the hippocampus) 6 h after injection, and a decrease (bleaching) in some terminal fields starting 12 h after injection and increasing at 24–48 h. The areas most affected by the zinc bleaching were the olfactory bulb, piriform and entorhinal cortices, endopiriform and amygdaloid nuclei. Transitory Fos immunostaining (within neuronal nuclei) was observed between 3 and 12 h after kainate treatment in many telencephalic areas: olfactory bulb, cortex (piriform, hippocampal and neocortex) and amygdaloid nuclei. This was accompanied by changes in glial markers starting 3 h after injection. Fluoro-Jade B staining in neurones (degeneration) appeared 6 h after treatment and increased later. Degenerating areas generally coincided with those showing Fos immunoreactivity. Zinquin and sulphide/silver methods revealed various pools of zinc after kainate injection: a cytoplasmic pool and a terminal field (or vesicular) pool. Cytoplasmic zinc (zinquin) was coincident, in time and location, with cell degeneration, thus implicating zinc in cell death. This zinc may not have come from presynaptic stores, since no bleaching (sulphide/silver method) was observed 6 h after injection. Future experiments altering zinc pools (e.g. by chelating agents) may elucidate the function of zinc.

Translocation of Glutamate Transporter Subtype Excitatory Amino Acid Carrier 1 Protein in Kainic Acid-Induced Rat Epilepsy

Glutamate excitotoxicity has been implicated in the pathophysiology of epilepsy. Systemic injection of kainic acid (KA) in the rat produces an animal model of human temporal lobe epilepsy. We examined the temporal expression of the sodium-dependent neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), in KA-induced rat epilepsy. As an early alteration, perinuclear deposits of EAAC1 protein were found mainly in the large pyramidal neurons at the hippocampus, neocortex, piriform cortex, and amygdala with the reduction of neuropil staining 6 hours after KA injection. Immunoelectron microscopic study revealed that the perinuclear EAAC1 immunoreactivity corresponded to the translocation to the Golgi complex. At this time point, EAAC1 mRNA was down-regulated. The intracellular aggregation of EAAC1 primarily disappeared by 24 hours. In vitro studies indicated that internalization of EAAC1 from the plasma membrane to the intracellular compartment by KA treatment was associated with the reduction of electrogenic transporter currents. Our results suggest that the transient EAAC1 internalization participates in the modulation of the transporter function preventing excessive glutamate uptake to pyramidal neurons during the early stage of epilepsy.