The effects of ethanol on the hippocampal neural tissue development and kainite receptor expression in young mouse / 中国神经精神疾病杂志

Objectives To investigate the effects of ethanol on neural development and kainate receptor expression in young mice. Methods Fetal alcohol spectrum disorder model was established by administration of 20% ethanol solu?tion to 7-day-old Kunming mice and control animals received physiological saline (The number of treatment and control were 80 and 40, respectively ). Body weight and general biological features were observed every day. Morris water maze was used to test learning and memory ability. Fluoro-Jade B was used to examine neural cells 24 hours after treatment in additional thirty 7-day-old Kunming mice which were further divided into two groups:a treatment group receiving 20%ethanol solution (n=15) and a control group receiving physiological saline (n=15). The development of neural cells and expression levels of kainite receptors were examined by using immunofluorescence staining. Results The body weight was significantly lighter in treatment group than in control group(control:21.13 ± 1.72g,treatment:13.96 ± 2.98g,P<0.05). Morris test showed that model group had longer latency than control group to find hidden platform(control:21.05± 5.31s,treatment:34.15±3.26s,P<0.05). Spatial probe test revealed that the number of passing through the platform were significantly smaller in model group than in control group(control:2.70 ± 1.25 times,treatment:0.93 ± 0.80 times,P<0.05). Astrocyte development anomaly was evident after ethanol treatment for 7 days. The expression levels of kainite re?ceptor GluR-6 and KA2 were up-regulated in the CA region of the hippocampus after ethanol treatment for 7 days. Con?clusion Kainite receptor GluR-6 and KA2 in CA region of the hippocampus may contribute to ethanol-induced hippo?campal neural development anomaly.

Role of glutamate receptor-6 in kainate-induced epilepsy in rats / 西安交通大学学报(医学版)

Objective To study the molecular mechanism of glutamate receptor-6 (GluR6) in the pathogenesis of epilepsy. Methods Seizure model of SD rats was induced by intraperitoneal injection of kainate (KA). Immunoprecipitation and immunoblotting were performed to examine the interactions of GluR6 and MLK3 with PSD95 at various time points after KA injection. The effect of Tat-GluR6-9c on the MLK3 phospharylation induced by kainate was observed with immunoblotting and immunohistochemistry. Results The assembly of GluR6 and MLK3 with PSD95 was induced after KA hippocampal CA3 region, and bagan to decrease one day later. Pretreatment after KA injection in CA3 region (P<0.05). Conclusion KA induces the assembly of the GluR6-PSD95-MLK3 signaling module and subsequently activates MLK3, which ultimately results in brain injury.

Type II and III Taste Bud Cells Preferentially Expressed Kainate Glutamate Receptors in Rats

Glutamate-induced cobalt uptake reveals that non-NMDA glutamate receptors (GluRs) are present in rat taste bud cells. Previous studies involving glutamate induced cobalt staining suggest this uptake mainly occurs via kainate type GluRs. It is not known which of the 4 types of taste bud cells express subunits of kainate GluR. Circumvallate and foliate papillae of Sprague-Dawley rats (45~60 days old) were used to search for the mRNAs of subunits of non-NMDA GluRs using RT-PCR with specific primers for GluR1-7, KA1 and KA2. We also performed RT-PCR for GluR5, KA1, PLCbeta2, and NCAM/SNAP 25 in isolated single cells from taste buds. Taste epithelium, including circumvallate or foliate papilla, express mRNAs of GluR5 and KA1. However, non-taste tongue epithelium expresses no subunits of non-NMDA GluRs. Isolated single cell RT-PCR reveals that the mRNAs of GluR5 and KA1 are preferentially expressed in Type II and Type III cells over Type I cells.

Effects of NMDA, AMPA and Kainate on the Release of Acetylcholine in Rat Hippocampal and Striatal Slices

This study examined the effects of N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate on basal and electrically-evoked release of acetylcholine (ACh) from the rat hippocampal and striatal slices which were preincubated with [3H]choline. Unexpectedly, the basal and evoked ACh release were not affected at all by the treatment with NMDA (3~100microM), AMPA (1~100microM) or kainate (1~100microM) in hippocampal slices. However, in striatal slices, under the Mg2 -free medium, 30microM NMDA increased the basal ACh release with significant decrease of the electrically- evoked releases. The treatment with 1microM MK-801 not only reversed the 30microM NMDA-induced decrease of the evoked ACh release, but also attenuated the facilitatory effect of 30microM NMDA on the basal ACh release. The treatment with either 30microM AMPA or 100microM kainate increased the basal ACh release without any effects on the evoked release. The treatment with 10microM NBQX abolished the AMPA- or kainate-induced increase of the basal ACh release. Interestingly, NBQX significantly attenuated the evoked release when it was treated with AMPA, although it did not affect the evoked release alone without AMPA. These observations demonstrate that in hippocampal slices, ionotropic glutamate receptors do not modulate the ACh release in cholinergic terminals, whereas in striatal slices, activations of ionotropic glutamate receptors increase the basal ACh release though NMDA may decrease the electrically-evoked ACh release.

Rapid Loss of Apurinic/Apyrimidinic Endonuclease and Subsequent Apoptosis in Kainate-Induced Seizure Model / 대한간질학회지

PURPOSE: The DNA repair enzyme, apurinic/apyrimidinic endonuclease (APE) plays a role in base excision repair pathway involved in repairing apurinic/apyrimidinic (AP) site after oxidative stress. To reveal the relationship between APE and neuronal apoptosis associated with oxidative stress after kainate treatment, the temporal change of APE expression was investigated in kainate-induced seizure model. METHODS: Status epilepticus was induced by unilateral intrahippocampal injection of kainate. Superoxide anion radical production and DNA oxidation were evaluated by in situ detection of oxidized hydroethidine and 8-hydroxyguanine (8-OHG) immunore activity. APE expression was examined by Western blot and immunohistochemical analysis. DNA fragmentation was visualized with terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining. RESULTS: Cell loss occurred at 24 hr in CA1, CA2, and CA3 after kainate-injection. 8-OHG immunoreactivity and oxidized hydroethidine were increased comparing with control after kainate-injection. APE immunoreactivity was decreased 4 and 24 hours in the hippocampus after kainate-injection. TUNEL-positive cells were observed 24 hours but not 4 hours in hippocampus after kainate-injection. In double labeling with APE and TUNEL, TUNEL-positive cells did not show APE immunoreactivity. These data showed that cellular oxidative stress was increased, thereby APE was decreased in the hippocampus after kainate-injection. Also, it was shown that the reduction of APE preceded DNA fragmentation. CONCLUSION: This study suggests that rapid loss of APE may produce the failure of DNA repair-machinary and then induce neuronal apoptosis following kainate-injection.

Seizure -Related Change of NADPH -diaphorase and Calcium Binding Protein Positive Neurons in the Brain of Rats / 체질인류학회지

Nitric oxide (NO) is a gaseous messenger that plays a role in neurotransmission, long term potentiation, depression and cerebral blood flow. Increases in intracellular calcium levels activate the enzyme NOS, and the NO released then diffuse to adjacent cells and activate guanylate cyclase. NO mediates the increase in cerebral blood flow during seizure activity. Therefore, the present study was aimed to investigate the change of NOS and calcium binding proteins in the rat cerebral cortex following seizure. Rats were injected with kainate (KA) and killed at 6 hours, 1, 3, 5 and 10 days after seizure. Expressional change of nNOS, calbindin D28k and parvalbumin was assessed by histochemistry, immunohistochemistry and microdensitometry in the rat brain. The intensity of the NADPH -d staining in rat cortical neurons showed a marked susceptibility to KA administration. At 6 hours and 3 days after seizure, the optical density of the NADPH -d staining was increased relative to the signal in saline treated control rats. At 5 and 10 days after seizure, the optical density of NADPH -d staining was not significantly different in most cortical regions compared to controls. In the hippocampus, the optical density of NADPH -d staining was highest at 5 days after seizure. The optical densities of calbindin D28k and parvalbumin positive neurons were various in the cerebral cortex, hippocampus and caudatoputamen during postseizure period. These results indicate that the calcium binding proteins investigated here are not essential for determining the activation of nNOS/NADPH -d positive neurons in the cerebral cortex and striatum.

Zn2+ modulates the responses of rat dorsal horn neuron to C-fiber stimulation and excitatory amino acids

Zinc contained in the neurons of central nervous system is activity-dependently released and then attenuates NMDA (N-methyl-D-aspartate)-induced neurotoxicity while augmenting non-NMDA-induced neurodegeneration. Zinc also has been reported to produce antinociceptive action on the inflammation- and nerve injury-induced hyperalgesia in the behavioral test. In this study, we investigated the effects of zinc on the responses of dorsal horn cells to NMDA, kainate and graded electrical stimulation of C-fibers. In the majority of WDR cells (70.6%), zinc current-dependently inhibited WDR cell responses to NMDA and in the remaining cells, produced biphasic responses; excitation followed by inhibition. Zinc augmented the responses of WDR cells to iontophoretical application of kainate. The dominant effect of Zn2+ on the responses of WDR cells to C-fiber stimulation was excitatory, but inhibition, excitation-inhibition and no change of the responses to C-fiber stimulation were induced. Ca2+-EDTA antagonized the excitatory or inhibitory effects of Zn2+ on the WDR cell responses. These experimental findings suggest that Zn2+ modulates the transmission of sensory information in the rat spinal cord.

Zinc translocation and heat shock protein induction in rat brains following kainate seizures / 대한해부학회지

Translocation of synaptic zinc may mediate neuronal death in pathological conditions. In this study, we examined the possible correlation between zinc translocation and heat shock protein (HSP)72 induction in rat brains following kainate seizures. Zinc accumulation, visualized by Timm's method, occurred in degenerating neurons in hippocampus, amygdala, and cortex 6~24 h after kainate injection. Immunohistochemistry with anti-HSP72 antibody revealed HSP induction largely in areas where zinc accumulation occurred. At the cellular level, however, most HSP72 immunoreac-tive neurons were found to be Timm (-) and morphologically intact. Present results suggest that intense zinc translocation may induce neuronal death before possible HSP induction. However, we could not rule out the possibility that sublethal zinc translocation, below the detection limit by Timm's method, may play a role in HSP72 induction.

Distribution and Expression of Kainate(KA) Receptor Subunits in Moderate Hypoxic Newborn Piglet Brain

PURPOSE: The mechanism of hypoxic damage is mainly intracellular influx of calcium ions through the glutamate ionotropic receptor. This study was performed to determine alterations in distribution and expression of kainate receptor subunits after 1 hour of moderate hypoxia in the newborn piglet brain, as in a condition of mild to moderate perinatal hypoxic-ischemic encephalopathy. METHODS: Ten newborn piglets were ventilated at PaO2 over 80mmHg for 30min. Thereafter, the control group(n=5) was ventilated with 21% oxygen, and hypoxic group(n=5) with 6% oxygen at PaO2 below 25mmHg for 1 hour. Concentrations of protein, ATP and phosphocreatine were determined. The proteins were immunostained with anti-rat GluR6/7 and anti-rat KA2 antibody. RESULTS: Hypoxia(PaO2 20+/-1mmHg) and acidosis(pH 7.06+/-0.09) developed significantly in the hypoxic group compared to the control group(PaO2 104+/-4mmHg, pH 7.44+/-0.03, respectively, Phippocampus, thalamus, hypothalamus>basal ganglia, cerebellum>white matter, and KA2 subunits were ordered : hippocampus, basal ganglia>cerebral cortex>thalamus, cerebellum>hypothalamus, white matter. The distribution of the subunits between the hypoxic group and control group were similar. CONCLUSION: Cerebral cortex, hippocampus and basal ganglia may be the most vulnerable to excitotoxic injury. Kainate receptor subunits did not change after 1 hour of moderate hypoxia.

Distribution and Expression of Kainate(KA) Receptor Subunits in Moderate Hypoxic Newborn Piglet Brain

PURPOSE: The mechanism of hypoxic damage is mainly intracellular influx of calcium ions through the glutamate ionotropic receptor. This study was performed to determine alterations in distribution and expression of kainate receptor subunits after 1 hour of moderate hypoxia in the newborn piglet brain, as in a condition of mild to moderate perinatal hypoxic-ischemic encephalopathy. METHODS: Ten newborn piglets were ventilated at PaO2 over 80mmHg for 30min. Thereafter, the control group(n=5) was ventilated with 21% oxygen, and hypoxic group(n=5) with 6% oxygen at PaO2 below 25mmHg for 1 hour. Concentrations of protein, ATP and phosphocreatine were determined. The proteins were immunostained with anti-rat GluR6/7 and anti-rat KA2 antibody. RESULTS: Hypoxia(PaO2 20+/-1mmHg) and acidosis(pH 7.06+/-0.09) developed significantly in the hypoxic group compared to the control group(PaO2 104+/-4mmHg, pH 7.44+/-0.03, respectively, Phippocampus, thalamus, hypothalamus>basal ganglia, cerebellum>white matter, and KA2 subunits were ordered : hippocampus, basal ganglia>cerebral cortex>thalamus, cerebellum>hypothalamus, white matter. The distribution of the subunits between the hypoxic group and control group were similar. CONCLUSION: Cerebral cortex, hippocampus and basal ganglia may be the most vulnerable to excitotoxic injury. Kainate receptor subunits did not change after 1 hour of moderate hypoxia.

BDNF mRNA Expression and Calcium Influx Pathways in Kainate-induced Neurotoxicity

Kainate is known as a neurotoxin acting on the glutamate receptors in the central nervous system (CNS). Glutamate acts an excitatory neurotransmitter at physiological concentration but has a neurotoxic effect in excess amount. BDNF (brain-derived neurotrophic factor) has been reported to have a protective effect against the cellular toxicity and to plays an important role in neuronal survival and differentiation in peripheral nervous system. However, the functional mechanism of BDNF in CNS is unclear. This study was performed to examine the protective effect of BDNF in kainate-induced neurotoxicity and to observe the relation between BDNF mRNA expression and increasing pathways of intracellular Ca2+ concentration. Cultured hippocampal neurons were prepared from 17-18 day embryonic rats and used at the 7th day after neuronal culture. The amounts of BDNF mRNA were measured by reverse transcription polymerase chain reaction after the treatment of several glutamate receptor agonists: glutamate, kainate, -amino-3-hydroxyl-4-isoxazolepropionic acid, N-methyl-D-aspartate. Kainate showed the most prominent effect in an increase of BDNF mRNA expression among the glutamate receptor agonists. The maximal increase of BDNF mRNA expression was obtained in 50 M kainate at 3 hr after the treatment. Adding BDNF to kainate containing cultured hippocampal neurons diminished the increasing level of lactic dehydrogenase according to the single treatment of kainate. In the experiment to evaluate the Ca2+ influx pathways related in BDNF mRNA expression, nifedipine (10 M), a voltage-dependent Ca2+ channel blocker, decreased the both kainate (50 M) and KCl (50 mM) induced BDNF mRNA expressions by 18.4% and 35.0%, respectively. Ryanodine (10 M), a blocker of intracellular release from Ca2+ storage, however, did not show any effect in the both kainate- and KCl-treated neurons.These results suggest that BDNF has a protecting effect against the kainate-induced neurotoxicity in cultured rat hippocampal neurons, and its expression is more related with the Ca2+ influx through the voltage-dependent Ca2+ channels than the release from intracellular Ca2+ storage.

Effect of MK801 and CNQX on Retinal Injury Induced by Ischemia, NMDA, or Kainate

To examine the protection of retinal cell death by glutamate antagonists in vivo, this study was carried out in pressure-induced ischemia model. Firstly, we observed that ischemia resulted in the similar retinaldamage to the injuries caused by NMAD and Kainate toxicity. Secondly, the retinal cell death caused by ischemia was prevented by MK801 and CNQX, glutamate antagonists for NMDA and Kainate excitotoxicity, respectively at 24hr after ischemia. MK801 was shown to prevent the cell death in ganglion cell layer and CNQX in inner unclear layer. In addition, the combination of CNQX and MK801 protected the retina neuronal cell from ischemic injury better than when they were applied separately. The partial protection of retinal cell death by glutamate antagonists in ischemia model indicates that glutamate eoxicity as well as other cell death mechanism such as apoptosis mediates ischemia induced retinal cell death. Thus, cell death by other mechanism must be also blocked in order to prevent retinal cell death, completely.

c-kit and Ligand Expression in the Mouse Hippocampus after Kainate-induced Seizure / 대한해부학회지

In the present study, we have etamined the role of c-kit and KL ligand in the mouse brain after kainate-induced seizure. To investigate whether c-kit receptor and KL ligand might involved in kainate-induced apoptosis, the expression patterns of c-kit and KL mRNA and localization of immunoreactivity for c-Kit, SCF and Bcl-2 protein were examined by in situ hybridization technique and immunohistochemical method, respectively, in the mouse hippocampus after kainate treatment. This report is the first demonstration for the role of c-kit receptor and KL ligand in the kainate-induced apoptosis. Our conclusion is based on : 1] c-kit and KL mRNA expressions were increased in CA3 region of the hippocampus in 1h after kainate treatment, 2] immunoreactivities for c-Kit protein and SCF were detected higher level in the CA1 and CA3 sectors in 24h after kainate treatment, 3] expression level for Bcl-2 protein was increased in the CA3 region of the hippocampus 24h after kainate treatment. These results suggest that bcl-2 could promote cell survival of injured neurons in CA3 after kainate-induced seizure. And increased translations of c-kit receptor and KL ligand after kainate injection in this area susgest that c-kit receptor and KL ligand could have a role in the kainate-induced apoptosis.

Excitotoxic Cell Death in Cultured Retinal Neurons

We examined excitotoxicity, putatively a major mechanism of ischemic neuronal death, in primary rat retinal cultures. Retinal cultures were prepared from newborn rats (day 1 or 2). Exposure of these cultures (DIV8-10)to NMDA or kainate induced neuronal death. Furthermore, MK-801 or CNQX each partially attenuated glutamateinduced neuronal death, suggesting that both NMDA and kainate receptors mediate it. Thy-1(+) retinal ganglion neurons, like neurons as a whole, were equally injured by NMDA and by kainate. However, GABA(+) or calbindin (+) neurons of the inner nuclear layer were resistant to NMDA, but highly vulnerable to kainate. These neurons may have AMPA/kainate receptors that are highly permeable to Ca2+, as they take up cobalt with kainate stimulation. These results suggest that the AMPA/kainate receptor, rater than the NMDA receptor, may mediate this pattern of selective neurnonal death.

Experimental Study of Initiation of Cortical Spreading Depression by Excitatory Amino Acids Using a New Topical Application Model

The purpose of this study was, first, to devise a new model for topical application of excitatory amino acids(EAAs) to rat cerebral cortex that successfully and repeatdly initiate the cortical spreading depression(CSD). Then, by using this model, six major EAAs that are known to act on single or multiple subtypes of EAA receptor were examined; glutamate, kainate, aspartate, N-methyl-D-aspartate(NMDA), quisqualate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazoie-proprite(AMPA). Through the model, with a cone-shaped well buried in 1.5mm depth of the cerebral cortex, these chemical agents were topically applied to the cortical gray matter. A total of 50 Sprague-Dawley rats were used and divided into seven groups including the sham group. Doses of each EAA between 10(-7) and 10(-4)M concentrations were escalated for triggering the CSD and its rate of consistency in triggering was also evaluated. In the overall results. CSDs were repeatedly initiated in all experimental groups with relatively consistent rates. Duration of CSDs were 1-4 minutes(mean 2.2+/-1.4) and amplitudes were 20-40mV. Effective dose(50)(ED(50)), that trigger over 50% of CSD was 10(-5)M(n=8) for glutamate, 10(-7)M(n=8) for aspartate, 10(-5)M(n=7) for AMPA, 10(-5)M(n=7) for quisqualate, and 10(-4)M(n=7) for NMDA and kainate group. Among those acting on the single receptor, AMPA was shown to be the most effective in triggering CSD, and NMDA, and kainate were in descending orders. Aspartate that was known to act on multiple EAA receptors, showed the highest rate of triggering CSD among all groups, but glutamate, known to act on all receptors of its subtypes, showed the most consistent rate of triggering CSD at dose escalation. These results revealed that those EAA acting on multiple receptors, namely aspartate and glutamate, showed the highest and most consistent rate of triggering CSD. Among those acting on single channel of receptors. AMPA was the most effective, although its consistency and rate of triggering of CSD was somewhat lower than.

Effect of Topiramate on Spatial Learning and Memory of KA-induced Rats / 中国心理卫生杂志

Objective:To investigate the chronic effect of topiramate on spatial learning and memory to young rats. Methods:P28 KA-induced rats were used. After 8-week treatment of topiramate, spontaneous recurrent seizures were recorded; Spatial learning and memory ability was evaluated by Morris water maze. Results: TPM-treated rats had significantly fewer(3.50?3.84) spontaneous recurrent seizures than rats without TPM treatment(7.36?3.75). On the first day of Morris water maze, they also had statistically longer latency to the platform(590.6?230.9 s) than those from the group without TPM treatment (422.6?122.3 s), but no latency differences were found in the following three days. TPM had no effect on water maze performance to rats without KA induction. After three days of interval, no differences were found in all groups under the same experimental situation.Conclusion:For developing rat brain, TPM has no impairment on the ability of long-term memory and information retrieval meanwhile its negative effect on spatial learning is temporary.

EXPRESSION OF FOS-LIKE PROTEIN IN BRAIN FOLLOWING VAGUS NERVE STIMULATION IN EPILEPTIC RATS / 解剖学报

Objective To investigate the related brain areas and nucleus involved in the inhibition of vagus nerve stimulation (VNS) on epilepsy. Methods Using the kainic acid kindling epilepsy rats model,we observed the distribution of Fos positive neurons in the brain after VNS treatment combined with immunohistochemical method. Results VNS induced a significant increase in Fos immunoreactivity in the bilateral nucleus of solitary tract,the locus coeruleus,parabrachial nucleus,periaqueductal gray of midbrain,lateral habenular nucleus,paraventricular thalamic nucleus,rhomoid thalamic nucleus,paraventricular hypothalamic nucleus.Dense Fos immunoreactive staining was also seen in the central nucleus of amygdala,bed nucleus of stria terminalis,lateral septal nucleus and prepirifiorm cortex.Pretreatment with electric stimulation on cervical vagual nerve stem, c fos expressing of hippocampus formation,cingulate gyrus and frontal,parietal,temporal lobus significantly diminished after KA injection. Conclusion This finding may suggest that VNS activates various brain structure that could be involved in the regulation of seizures.