Systemic Injection Of Kainate Research Articles

Seizure-induced increase in microglial cell population in the developing zebrafish brain

Epilepsy is a chronic brain disorder characterized by unprovoked and recurrent seizures, of which 60% are of unknown etiology. Recent studies implicate microglia in the pathophysiology of epilepsy. However, their role in this process, in particular following early-life seizures, remains poorly understood due in part to the lack of suitable experimental models allowing the in vivo imaging of microglial activity. Given the advantage of zebrafish larvae for minimally-invasive imaging approaches, we sought for the first time to describe the microglial responses after acute seizures in two different zebrafish larval models: a chemically-induced epileptic model by the systemic injection of kainate at 3 days post-fertilization, and the didys552 genetic epilepsy model, which carries a mutation in scn1lab that leads to spontaneous epileptiform discharges. Kainate-treated larvae exhibited transient brain damage as shown by increased numbers of apoptotic nuclei as early as one day post-injection, which was followed by an increase in the number of microglia in the brain. A similar microglial phenotype was also observed in didys552-/- mutants, suggesting that microglia numbers change in response to seizure-like activity in the brain. Interestingly, kainate-treated larvae also displayed a decreased seizure threshold towards subsequent pentylenetetrazole-induced seizures, as shown by higher locomotor and encephalographic activity in comparison with vehicle-injected larvae. These results are comparable to kainate-induced rodent seizure models and suggest the suitability of these zebrafish seizure models for future studies, in particular to elucidate the links between epileptogenesis and microglial dynamic changes after seizure induction in the developing brain, and to understand how these modulate seizure susceptibility.

P.041 Magnesium and calcium reduce severity of spatial memory impairments in kainate mouse model of mesial temporal lobe epilepsy

Background: Calcium (Ca) and magnesium (Mg) are crucial in metabolism, excitability and neuroglial plasticity. Our aim was to evaluate whether Mg (20 mg/kg) or Ca (100 mg/kg) could improve the memory prognosis in the kainic model of mesial temporal epilepsy. Methods: Seizures were induced by systemic injection of kainate (8mg/kg) and mice were then treated by ions every 48 hours. A placebo (physiological solution) replaced kainate or ions in specific groups. Six cohorts were studied for seven weeks: control group (G0: no kainate and no ion, only placebo); untreated reference group (GR: kainate and then placebo); G1 groups were treated from the third day (G1m, G1c: kainate and then Mg/Ca); G2 groups were treated from the third week (G2m, G2c: kainate and then Mg/Ca). Radial maze and a classic maze were used for cognition evaluation. Results: The memory (short/long term) was differently affected by kainate or improved by Mg/Ca. The treated groups performed better than GR mice, but Mg was more effective. In addition, Mg demonstrated an increasing therapeutic effect over time while Ca showed an acute and apparently decreasing action in the G1c group. Conclusions: Mg should be considered for a clinical evaluation of its effect on epileptic disorders.

Regional and temporal regulation and role of somatostatin receptor subtypes in the mouse brain following systemic kainate-induced acute seizures

Somatostatin reduces neuronal excitability via somatostatin receptors (Sst1-Sst5) and inhibits seizure activity. However, the expression status of the Sst subtypes in epileptic mice and their role in the antiepileptic effects of somatostatin remain unclear. Here, we show that the Sst subtypes are regulated differently by epileptic neuronal activity in mice. Systemic kainate injection rapidly and transiently elevated the Sst2 and Sst3 mRNA and reduced Sst1 and Sst4 mRNA in the hippocampus; however, among all the subtypes, only Sst2 mRNA was increased in the excitatory neurons of the basolateral amygdala, accompanied by a decrease in the level of Sst2 protein. Following kainate administration, recovery from seizure was delayed by reduced expression of Sst2 in the basolateral amygdala, but not in the dentate gyrus of the hippocampus; higher expression levels of Bdnf, a neuronal activity marker, were observed in both conditions. These results suggest that Sst2 contributes to seizure termination by feedback inhibition in the amygdala. This could be a potential therapeutic target for acute seizures.

New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein.

Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3’UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future.

Predominant Functional Expression of Kv1.3 by Activated Microglia of the Hippocampus after Status epilepticus

BackgroundGrowing evidence indicates that the functional state of microglial cells differs according to the pathological conditions that trigger their activation. In particular, activated microglial cells can express sets of Kv subunits which sustain delayed rectifying potassium currents (Kdr) and modulate differently microglia proliferation and ability to release mediators. We recently reported that hippocampal microglia is in a particular activation state after a status epilepticus (SE) and the present study aimed at identifying which of the Kv channels are functionally expressed by microglia in this model.Methodology/Principal FindingsSE was induced by systemic injection of kainate in CX3CR1eGFP/+ mice and whole cell recordings of fluorescent microglia were performed in acute hippocampal slices prepared 48 h after SE. Microglia expressed Kdr currents which were characterized by a potential of half-maximal activation near −25 mV, prominent steady-state and cumulative inactivations. Kdr currents were almost abolished by the broad spectrum antagonist 4-Aminopyridine (1 mM). In contrast, tetraethylammonium (TEA) at a concentration of 1 mM, known to block Kv3.1, Kv1.1 and 1.2 subunits, only weakly reduced Kdr currents. However, at a concentration of 5 mM which should also affect Kv1.3 and 1.6, TEA inhibited about 30% of the Kdr conductance. Alpha-dendrotoxin, which selectively inhibits Kv1.1, 1.2 and 1.6, reduced only weakly Kdr currents, indicating that channels formed by homomeric assemblies of these subunits are not important contributors of Kdr currents. Finally, agitoxin-2 and margatoxin strongly inhibited the current.Conclusions/SignificanceThese results indicate that Kv1.3 containing channels predominantly determined Kdr currents in activated microglia after SE.

Somatostatin receptor subtype 4 couples to the M-current to regulate seizures.

The K(+) M-current (I(M), Kv7) is an important regulator of cortical excitability, and mutations in these channels cause a seizure disorder in humans. The neuropeptide somatostatin (SST), which has antiepileptic properties, augments I(M) in hippocampal CA1 pyramidal neurons. We used SST receptor knock-out mice and subtype-selective ligands to investigate the receptor subtype that couples to I(M) and mediates the antiepileptic effects of SST. Using pentylenetetrazole as a chemoconvulsant, SST(2), SST(3), and SST(4) receptor knock-out mice all had shorter latencies to different seizure stages and increased seizure severity when compared with wild-type mice. However, the most robust differences were observed in the SST(4) knock-outs. When seizures were induced by systemic injection of kainate, only SST(4) knock-outs showed an increase in seizure sensitivity. We next examined the action of SST and subtype-selective SST agonists on electrophysiological parameters in hippocampal slices of wild-type and receptor knock-out mice. SST(2) and SST(4) appear to mediate the majority of SST inhibition of epileptiform activity in CA1. SST lacked presynaptic effects in mouse CA1, in contrast to our previous findings in rat. SST increased I(M) in CA1 pyramidal neurons of wild-type and SST(2) knock-out mice, but not SST(4) knock-out mice. Using M-channel blockers, we found that SST(4) coupling to M-channels is critical to its inhibition of epileptiform activity. This is the first demonstration of an endogenous enhancer of I(M) that is important in controlling seizure activity. SST(4) receptors could therefore be an important novel target for developing new antiepileptic and antiepileptogenic drugs.

Induction of the Wnt Inhibitor, Dickkopf‐1, Is Associated with Neurodegeneration Related to Temporal Lobe Epilepsy

Inhibition of the Wnt pathway by the secreted glycoprotein, Dickkopf-1 (Dkk-1) has been related to processes of excitotoxic and ischemic neuronal death. We now report that Dkk-1 is induced in neurons of the rat olfactory cortex and hippocampus degenerating in response to seizures produced by systemic injection of kainate (12 mg/kg, i.p.). There was a tight correlation between Dkk-1 expression and neuronal death in both regions, as shown by the different expression profiles in animals classified as "high" and "low" responders to kainate. For example, no induction of Dkk-1 was detected in the hippocampus of low responder rats, in which seizures did not cause neuronal loss. Induction of Dkk-1 always anticipated neuronal death and was associated with a reduction in nuclear levels of beta-catenin, which reflects an ongoing inhibition of the canonical Wnt pathway. Intracerebroventricular injections of Dkk-1 antisense oligonucleotides (12 nmol/2 microL) substantially reduced kainate-induced neuronal damage, as did a pretreatment with lithium ions (1 mEq/kg, i.p.), which rescue the Wnt pathway by acting downstream of the Dkk-1 blockade. Taken collectively, these data suggest that an early inhibition of the Wnt pathway by Dkk-1 contributes to neuronal damage associated with temporal lobe epilepsy. We also examined Dkk-1 expression in the hippocampus of epileptic patients and their controls. A strong Dkk-1 immunolabeling was found in six bioptic samples and in one autoptic sample from patients with mesial temporal lobe epilepsy associated with hippocampal sclerosis. Dkk-1 expression was undetectable or very low in autoptic samples from nonepileptic patients or in bioptic samples from patients with complex partial seizures without neuronal loss and/or reactive gliosis in the hippocampus. Our data raise the attractive possibility that drugs able to rescue the canonical Wnt pathway, such as Dkk-1 antagonists or inhibitors of glycogen synthase kinase-3beta, reduce the development of hippocampal sclerosis in patients with temporal lobe epilepsy.

Flavonoid wogonin from medicinal herb is neuroprotective by inhibiting inflammatory activation of microglia.

Wogonin (5,7-dihydroxy-8-methoxyflavone), a flavonoid originated from the root of a medicinal herb Scutellaria baicalensis Georgi, has been previously shown to have anti-inflammatory activities in various cell types including macrophages. In this work, we have found that wogonin is a potent neuroprotector from natural source. Wogonin inhibited inflammatory activation of cultured brain microglia by diminishing lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta, and nitric oxide (NO) production. Wogonin inhibited NO production by suppressing inducible NO synthase (iNOS) induction and NF-kappaB activation in microglia. Inhibition of inflammatory activation of microglia by wogonin led to the reduction in microglial cytotoxicity toward cocultured PC12 cells, supporting a neuroprotective role for wogonin in vitro. The neuroprotective effect of wogonin was further demonstrated in vivo using two experimental brain injury models; transient global ischemia by four-vessel occlusion and excitotoxic injury by systemic kainate injection. In both animal models, wogonin conferred neuroprotection by attenuating the death of hippocampal neurons, and the neuroprotective effect was associated with inhibition of the inflammatory activation of microglia. Hippocampal induction of inflammatory mediators such as iNOS and TNF-alpha was reduced by wogonin in the global ischemia model, and microglial activation was markedly down-regulated by wogonin in the kainate injection model as judged by microglia-specific isolectin B4 staining. Taken together, our results indicate that wogonin exerts its neuroprotective effect by inhibiting microglial activation, which is a critical component of pathogenic inflammatory responses in neurodegenerative diseases. The current study emphasizes the importance of medicinal herbs and their constituents as an invaluable source for the development of novel neuroprotective drugs.

Selective calcification of rat brain lesions caused by systemic administration of kainic acid.

Dystrophic calcification of previously damaged areas of nervous tissue occurs in a wide range of human diseases. The relationship between astroglial and microglial reactions and deposits of calcium salts was studied for up to five months in rats with a brain lesion produced by systemic administration of kainate. The morphology and atomic composition of the calcium salt deposits was also studied. Two types of lesions, sclerotic and liquefactive, were observed. In sclerotic lesions hyperplasia and hypertrophy of astrocytes partially substituted for the lost neurons, reaching a maximum in about twenty-five days after treatment. In liquefactive lesions, the astrocytic reaction occurred only around the liquefactive area. Microglial reaction was similar in both types of lesion and reached its highest expression in about twenty-five days. Calcium deposits were observed in the sclerotic but not in the liquefactive lesions. Clearly distinguishable granules of calcium salts were observed in sclerotic lesions under scanning electron microscopy after only five days post-injection. The size of calcified granules increased with time reaching 40 micro m or more in diameter at five months. The atomic composition of these deposits, studied by X-ray microanalysis, showed a time-dependent increase in calcium concentration. While there was no clear relationship between astroglial and microglial reactions and calcium salt deposits, the systemic injection of kainate produced progressively larger and more concentrated calcium deposits in sclerotic, but not in liquefactive lesions.

Induction and Activation of Protein Kinase Cδ in Hippocampus and Cortex after Kainic Acid Treatment

Various isoforms of protein kinase C (PKC), especially the novel PKC subtypes δ, ε, and the atypical subtype PKCζ, are involved in delayed cell death. We studied the expression and late activation of the latter PKC isoforms in comparison with classic PKCα, β, and γ in the brains of rats exposed to systemic kainate injection. The expression of PKCδ mRNA was strikingly upregulated (13-fold) in the cortex and the CA1 and CA3 hippocampal regions on 1 day after kainate administration, whereas PKCζ mRNA was only moderately increased (about 100%) in these three brain regions on day 2 following the drug. PKCε mRNA was slightly increased only in the cortex on days 2 and 6, while the mRNA levels of the classic PKC subtypes (α, β, and γ) remained unchanged or decreased after the treatment. Immunoblotting analyses revealed that the level of PKCδ protein started to increase on day 1 after kainate and was significantly elevated on day 2 in both the membrane and cytosol fractions of cortex and hippocampus. PKCε protein only showed a marginal increase and the level of PKCζ protein remained unaltered in response to the treatment. Cortical and CA1-3 pyramidal neurons displayed strong immunoreactivity for PKCδ on days 1 and 2, and microglia on days 1, 2, and 4 after the drug. The results indicate that the expression of apoptosis-associated isoforms of PKC, most notably that of δ, but to lesser extent also that of ε and ζ, is increased during kainate-induced neuronal death. The predominant induction of PKCδ in neurons and microglia suggests that PKCδ could be the major mediator or modulator of apoptotic and inflammatory responses to excitotoxic insults.

Experience-Dependent Plasticity of Mouse Visual Cortex in the Absence of the Neuronal Activity-Dependent Markeregr1/zif268

Neuronal activity elicits a rapid increase in the expression of several immediate early genes (IEGs). To clarify a role for IEG response in activity-dependent development, we examined the contribution of the egr1/zif268 gene during visual cortical processing and plasticity in mice. We first analyzed the expression of egr1 mRNA in wild-type (WT) mice using Northern blot hybridization. In the visual cortex, expression of egr1 mRNA increased dramatically after eye opening, systemic injection of kainate, or 30 min of photostimulation after a brief (5 d) period of dark adaptation. Thus, the expression of egr1 is regulated by synaptic activity in the mouse visual cortex, as it is in other species (e.g., monkeys, cats, and rats). To evaluate whether this transcription factor is directly involved in activity-dependent plasticity, mice lacking Egr1 were deprived of the use of one eye during the developmental critical period [postnatal day 24 (P24)-P34]. Extracellular in vivo single-unit recordings from the binocular zone of the visual cortex revealed that visual responses developed normally in egr1 knock-out (KO) mice. Moreover, a similarly significant shift of responsiveness in favor of the open eye was produced in both KO and WT mice by either brief (4 d) or long-term (>2 weeks) occlusion of one eye. There was no apparent compensation among egr2, egr3, or c-fos mRNA and protein expression in the visual cortex of egr1 KO mice. Taken together, these results indicate that egr1 is a useful marker of sensory input in mice but is not intrinsically necessary for the experience-dependent plasticity of the visual cortex. Our findings underscore a mechanistic distinction between sensory plasticity and long-lasting forms of synaptic potentiation in the hippocampus, for which egr1/zif268 was recently found to be essential.

Cyclooxygenase-2 selective inhibitors aggravate kainic acid induced seizure and neuronal cell death in the hippocampus

Cyclooxygenase-2 (COX-2) in the brain is expressed constitutively and also increased in pathological conditions such as seizure, cerebral ischemia, and inflammation. This study examined the role of COX-2 in kainic acid-induced seizure and in the following neuronal death by using selective inhibitors. Systemic kainate injection (50 mg/kg; i.p.) in mice evoked seizure within 15 min and led to 29% mortality within 2 h. TUNEL-positive neuronal death peaked at 3 days after injection and was prominent in CA 3a regions of the hippocampus. NS-398 or celecoxib (10 mg/kg, COX-2 selective inhibitor) and indomethacin (5 mg/kg, nonselective inhibitor) exaggerated kainic acid-induced seizure activity and mortality. COX-2 selective inhibitors induced the seizure at earlier onset and more severe mortality within the first hour than indomethacin and aspirin. NS-398 also aggravated kainic acid-induced TUNEL positive neuronal death and decreased Cresyl violet stained viable neurons, and extended lesions to CA 1 and CA 3b. Kainic acid increased the levels of PGD 2, PGF 2a and PG E 2 in the hippocampus immediately after injection. Indomethacin attenuated the production of basal and kainic acid-induced prostaglandins. In contrast, NS-398 failed to reduce until the first 30 min after kainic acid injection, during which the animals were severely seizured. It has been challenged the endogenous PGs might have anticonvulsant properties. Thus, COX-2 selective inhibitor, including nonselective inhibitor such as indomethacin, aggravated kainic acid-induced seizure activity and the following hippocampal neuronal death even with variable prostaglandin levels.

Impact of neonatal kainate treatment on hippocampal insulin-like growth factor receptors

The insulin-like growth factors-I and -II have neurotrophic properties and act through specific membrane receptors. High levels of binding sites for these growth factors are distributed discretely throughout the brain, being concentrated in the hippocampal formation. Functionally, the insulin-like growth factors, in addition to their growth-promoting actions, are considered to play important roles in normal cell functions, as well as in response to pharmacological or surgical manipulations. In adult rats, we have previously shown that systemic injection of kainate produces an overall decrease, in a time-dependent manner, in insulin-like growth factor-I and -II receptor binding sites in the hippocampus [Kar S. et al. (1997) Neuroscience 80, 1041–1055]. Given the evidence that insulin-like growth factors play a critical role during the early stages of brain development, the present study is a logical extension of this earlier report and established the effect of neonatal kainate injection on the developmental profile of insulin-like growth factor receptors. We have evaluated the time-course alteration of these receptors following systemic injection of kainate to newborn rats. After injection of a sublethal dose of kainate (5 mg/kg, i.p.) to postnatal one-day-old pups, [ 125I]insulin-like growth factor-I, [ 125I]insulin-like growth factor-II and [ 125I]insulin binding sites were studied at different postnatal days (7, 14, 21, 28 and 35) using receptor autoradiography. In the developing hippocampus, insulin-like growth factor-I and insulin binding sites are concentrated primarily in the dentate gyrus and the CA2/CA3 subfields, whereas insulin-like growth factor-II binding is discretely localized to the pyramidal layer and the granular layer of the dentate gyrus. Following kainate injection, we observed a slight increase in insulin-like growth factor-I binding sites in given hippocampal subfields starting at postnatal day 14, being significant at day 21. At later days, a progressive decrease was noted. This transient increase may represent an attempt for neuronal plasticity by up-regulating receptor levels. In contrast, insulin-like growth factor-II and insulin receptor binding sites are found to be decreased in various regions of the hippocampus in kainate-treated pups. Taken together, these results provide further evidence for the existence and differential alterations of insulin-like growth factor-I, insulin-like growth factor-II and insulin receptors in the developing rat hippocampus following kainate-induced lesion, suggesting possible involvement of these growth factors in brain plasticity.

Aging-associated up-regulation of neuronal 5-lipoxygenase expression: putative role in neuronal vulnerability.

Aging is associated with neurodegenerative processes. 5-Lipoxygenase (5-LO), which is also expressed in neurons, is the key enzyme in the synthesis of leukotrienes, inflammatory eicosanoids that are capable of promoting neurodegeneration. We hypothesized that neuronal 5-LO expression can be up-regulated in aging and that this may increase the brain's vulnerability to neurodegeneration. We observed differences in the distribution of 5-LO-like immunoreactivity in various brain areas of adult young (2-month-old) vs. old (24-month-old) male rats. Greater 5-LO-like immunoreactivity was found in old vs. young rats, in particular in the dendrites of pyramidal neurons in limbic structures, including the hippocampus, and in layer V pyramidal cells of the frontoparietal cortex and their apical dendrites. The aging-increased expression of neuronal 5-LO protein appears to be due to increased 5-LO gene expression. Using a quantitative reverse transcription/polymerase chain reaction assay and 5-LO-specific oligonucleotide primers and their mutated internal standards, we observed about a 2.5-fold greater hippocampal 5-LO mRNA content in old rats. 5-LO-like immunoreactivity was also observed in small, nonpyramidal cells, which were positive for glutamic acid decarboxylase or glial fibrillary acid protein. This type of 5-LO immunostaining did not increase in the old rats. Hippocampal excitotoxic injury induced by systemic injection of kainate was greater in old rats. Neuroprotection was observed with the 5-LO inhibitor, caffeic acid. Together, these results suggest that aging increases both neuronal 5-LO expression and neuronal vulnerability to 5-LO inhibitor-sensitive excitotoxicity, and indicate that the 5-LO system might play a significant role in the pathobiology of aging-associated neurodegenerative diseases.

A single dose of kainic acid elevates the levels of enkephalins and activator protein-1 transcription factors in the hippocampus for up to 1 year.

Neuronal plasticity plays a very important role in brain adaptations to environmental stimuli, disease, and aging processes. The kainic acid model of temporal lobe epilepsy was used to study the long-term anatomical and biochemical changes in the hippocampus after seizures. Using Northern blot analysis, immunocytochemistry, and Western blot analysis, we have found a long-term elevation of the proconvulsive opioid peptide, enkephalin, in the rat hippocampus. We have also demonstrated that an activator protein-1 transcription factor, the 35-kDa fos-related antigen, can be induced and elevated for at least 1 year after kainate treatment. This study demonstrated that a single systemic injection of kainate produces almost permanent increases in the enkephalin and an activator protein-1 transcription factor, the 35-kDa fos-related antigen, in the rat hippocampus, and it is likely that these two events are closely associated with the molecular mechanisms of induction of long-lasting enhanced seizure susceptibility in the kainate-induced seizure model. The long-term expression of the proenkephalin mRNA and its peptides in the kainate-treated rat hippocampus also suggests an important role in the recurrent seizures of temporal lobe epilepsy.

Expression of the proto-oncogene bcl-2 is increased in the rat brain following kainate-induced seizures

The proto-oncogene bcl-2 is an important suppressor of apoptotic cell death in development and of both apoptotic and necrotic cell death in mature neurons. We studied expression of bcl-2 and the related gene, bax, which may promote cell death, after seizures induced by systemic kainate injection in rats. Expression of bcl-2 mRNA was studied by in situ hybridization. Bax and bcl-2 protein expression was studied by immunocytochemistry. Histologic analysis of cresyl violet-stained paraffin sections was performed at 72 h. bcl-2 protein was expressed in CA1 neurons, a region that is injured, yet survives after seizures. Bcl-2 mRNA was expressed in CA3, a region where there is extensive neuronal death at 72 h, but the bcl-2 protein was not translated. However, bax protein expression in CA3 was increased at 24 h. These results support a possible role for bcl-2 in promoting survival of CA3 after seizures.

Induction of heat-shock protein-70 messenger RNA and protein following systemic kainate injection in the rat: evidence of protein axonal transport.

Induction of heat-shock protein-70 was studied using in situ hybridization and immunohistochemistry in the adult rat at different times following intraperitoneal injection of kainate. Marked expression of heat-shock protein-70 messenger RNA was observed during the first 24 h, followed by residual signal at 48 h. Inducible heat-shock protein-70 protein was found in sensitive areas not subjected to early cell death, including the lateral, dorsal and cingular cortices, lateral amygdala, thalamus and hippocampus at 12, 24 and 48 h after injection. In the hippocampus, inducible heat-shock protein-70 immunoreactivity was contained in the soma and proximal dendritic region of neurons of CA3, hilus and CA1 at 12 h, and within the entire dendritic arbor at 24 h. Heat-shock protein-70 immunoreactivity decreased in the cell bodies at four days, but delicate immunostaining appeared in the dorsal fornix, fimbria, and ventral and dorsal hippocampal commissures, as well as in the strata oriens and radiatum of CA3, and part of the stratum radiatum of CA1. Inducible heat-shock protein-70 immunoreactivity at day 7 was mainly localized in the strata oriens and radiatum of CA1 and CA3, and inner one-third of the molecular layer of the dentate gyrus, in which the ipsilateral and commissural hippocampal pathways terminate. These findings show that, in the hippocampus, inducible heat-shock protein-70 is synthesized in the cytoplasm of neurons and subsequently transported at slow rates (about 2-5 mm/day) through the axons to appropriate terminals in the ipsilateral and contralateral hippocampus. A similar pattern is observed for sensitive neurons (heat-shock protein-70 immunoreactive) in the neocortex and thalamus, and labelling of corticocortical, corticostriatal and intrathalamic (between the dorsal and the reticular nuclei) fibres. Since inducible heat-shock protein-70 keeps native proteins unfolded to prevent abnormal configuration following diverse insults, heat-shock protein-70 is proposed as a marker of transient impaired assembly of native proteins in sensitive neurons and axons following intraperitoneal kainate injection.

Dynamic changes in the composition of the AP-1 transcription factor DNA-binding activity in rat brain following kainate-induced seizures and cell death.

Kainate, a potent excitatory and neurotoxic agent, has also proved useful in studies on other glutamate-driven phenomena, such as neuronal plasticity. Long-term effects of kainate are apparently dependent on its influence on the expression of various genes, including those encoding the AP-1 transcription factor, consisting of proteins belonging to the Fos and Jun families. In our studies we analysed c-fos, fos B, c-jun, jun B and jun D mRNA levels as well as a functional feature of AP-1, its DNA-binding activity, in the rat brain following systemic injection of kainate. Two phases of elevated AP-1 DNA-binding activity were observed in the hippocampus and entorhinal cortex, and were correlated with period of seizures (2 and 6 h after kainate injection) and neuron damage (48-72 h). At 72 h after kainate treatment DNA fragmentation, believed to be diagnostic of apoptotic processes typical of programmed cell death phenomena, was noted. Two and six hours after the treatment, AP-1 consisted predominantly of Fos B, c-Fos, Fra-2 and Jun B, while at 72 h Jun D constituted the major AP-1 component in place of Jun B, and no c-Fos was detected. Only a slight AP-1 increase was seen 24 h after kainate treatment. In the sensory cortex, only the late phase of AP-1 elevation was detected. Contrary to AP-1, no effect of kainate on levels of two other transcription factors, CREB/ATF (cAMP-responsive element binding proteins) and OCT (octamer element DNA-binding activity) was seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged expression of AP-1 transcription factors in the rat hippocampus after systemic kainate treatment

Systemic administration of kainate, a glutamate receptor agonist, caused neuronal death in the CA1 and CA3 fields of the rat hippocampus. In the areas of cell loss, reactive astrocytes increased their expression of an astrocyte-specific protein, glial fibrillary acidic protein (GFAP). AP-1 DNA binding activity and the expression of a 35 kDa fos-related antigen (fra) remained elevated in the rat hippocampus for at least 2 weeks after a single systemic injection of kainate, which correlated with changes in gene expression during reactive gliosis. Immunoreactivity for fras was detected in the nuclei of neurons in the dentate gyrus, but relatively few cells in CA1 and CA3 were immunoreactive 1 week after kainate treatment. However, elevated AP-1 DNA binding activity was observed in the CA1 and CA3 regions as well as in the dentate gyrus, suggesting that proteins other than the fras were involved in the astrocytic AP-1 complex. The AP-1 DNA binding activity in hippocampus recognized an AP-1 sequence from the promoter region of the GFAP gene, suggesting that GFAP is a potential target gene. Thus, a single systemic injection of kainate causes long-term activation of AP-1 DNA binding activity in the rat hippocampus and may be important for long-term changes in gene expression in hippocampal cells.

Effects of kainate-induced seizures on cerebral metabolism: a combined 1H and 31P NMR study in rat

The cerebral metabolic changes elicited by kainate-induced seizures in the rat were investigated by in vivo combined NMR spectroscopy of 31P and 1H. Systemic injection of kainate induced no significant changes in cerebral ATP or PCr levels during up to 90 min of continuous, generalised seizures, and the cerebral 31P spectra showed only a transient mild cerebral acidosis 30 min after kainate administration. In parallel with the changes in intracellular cerebral pH, the 1H spectra showed a significant increase in lactate, which remained elevated throughout the seizures. These findings indicate that oxidative metabolism does not completely match the increased glycolysis during seizures though the energy homeostasis is maintained. This suggests that oxidative metabolism has a limited capacity to satisfy the brain's energy needs during the kainate-induced seizures, but that the different pathways of energy production in the brain cells can overcome this limitation. Thus the brain damage associated with this experimental model of epilepsy is not due to extended major failure of the energy supply.