Differential subcellular redistribution of protein kinase C isozymes in the rat hippocampus induced by kainic acid

Protein kinase C (PKC) consists of a family of Ca2+/phospholipid-dependent isozymes that has been implicated in the delayed neurotoxic effects of glutamate in vitro. In the present study, we assessed the effect of the glutamate analogue kainic acid (KA) on the subcellular expression of PKC isozymes in the hippocampus (HPC) in the period preceding (0.5, 1.5, 12, and 24 h) and during (120 h) hippocampal necrosis using western blot analysis and PKC isozyme-specific antibodies. Before subcellular fractionation (cytosol + membrane), hippocampi were microdissected into "HPC" (fields CA1-CA3) and "dentate gyrus" (DG; granule cells + hilus) regions. Four general patterns of alterations in PKC isozyme expression/distribution were observed following KA treatment. The first pattern was a relative stability in expression following KA treatment and was most apparent for cytosol PKCalpha (HPC + DG) and membrane (HPC) and cytosol (DG) PKCbetaII. The second pattern, observed with PKCgamma and PKCepsilon, was characterized by an initial increase in expression in both membrane and cytosolic fractions before seizure activity (0.5 h) followed by a gradual decrease until significant reductions are observed by 120 h. The third pattern, exhibited by PKCdelta, involved an apparent translocation, increasing in the membrane and decreasing in the cytosol, followed by down-regulation in both fractions and subsequent recovery. The fourth pattern was observed with PKCzeta only and entailed a significant reduction in expression before and during limbic motor seizures followed by a dramatic fivefold increase in the membrane fraction during the period of hippocampal necrosis (120 h). Although these patterns did not segregate according to conventional PKC isozyme classifications, they do indicate dynamic isozyme-specific regulation by KA. The subcellular redistribution of PKC isozymes may contribute to the histopathological sequelae produced by KA in the hippocampus and may model the pathogenesis associated with diseases involving glutamate-induced neurotoxicity.     

Expression and subcellular localization of thymosin beta15 following kainic acid treatment in rat brain

Thymosin β15 (Tβ15) is a pleiotropic factor which exerts multiple roles in the development of nervous system and brain diseases. In this study, we found that the expressions of Tβ15 mRNA and protein were substantially increased in several brain regions including hippocampal formation and cerebral cortex, following kainic acid (KA)-evoked seizures in rat. Interestingly, a subset of cortex neurons exhibited nuclear Tβ15 immunoreactivity upon KA treatment. Furthermore, translocation of Tβ15 from cytosol to nuclei was observed in cultured neurons or HeLa cells during staurosporine (STS)-induced apoptosis, which was also verified by time-lapse imaging of YFP-tagged Tβ15. It appeared that localization of Tβ15 is restricted to the cytosol in normal condition by its G-actin-interacting domain, because site-directed mutagenesis of this region resulted in the nuclear localization of Tβ15 in the absence of STS treatment. To explore the role of nuclear Tβ15, we enforced Tβ15 to localize in the nuclei by fusion of Tβ15 with nuclear localization signal (NLS-Tβ15). However, overexpression of NLS-Tβ15 did not alter the viability of cells in response to STS treatment. Collectively, these results suggest that nuclear localization of Tβ15 is a controlled process during KA or STS stimulation, although its functional significance is yet to be clarified.     

Neuroprotective effects of lactation against kainic acid treatment in the dorsal hippocampus of the rat

Marked hippocampal changes in response to excitatory amino acid agonists occur during pregnancy (e.g. decreased frequency in spontaneous recurrent seizures in rats with KA lesions of the hippocampus) and lactation (e.g. reduced c-Fos expression in response to N-methyl-d,l-aspartic acid but not to kainic acid). In this study, the possibility that lactation protects against the excitotoxic damage induced by KA in hippocampal areas was explored. We compared cell damage induced 24 h after a single systemic administration of KA (5 or 7.5 mg/kg bw) in regions CA1, CA3, and CA4 of the dorsal hippocampus of rats in the final week of lactation to that in diestrus phase. To determine cellular damage in a rostro-caudal segment of the dorsal hippocampus, we used NISSL and Fluorojade staining, immunohistochemistry for active caspase-3 and TUNEL, and we observed that the KA treatment provoked a significant loss of neurons in diestrus rats, principally in the pyramidal cells of CA1 region. In contrast, in lactating rats, pyramidal neurons from CA1, CA3, and CA4 in the dorsal hippocampus were significantly protected against KA-induced neuronal damage, indicating that lactation may be a natural model of neuroprotection.     

Role of α-CGRP in the regulation of neurotoxic responses induced by kainic acid in mice

Kainic acid (KA) is an excitatory and neurotoxic substance. The role of α-calcitonin gene-related peptide (α-CGRP) in the regulation of KA-induced hippocampal neuronal cell death was investigated in the present study. The intracerebroventricular (i.c.v.) administration with KA (0.07 μg) increased hippocampal α-CGRP mRNA level in ICR mice. The α-CGRP mRNA level began to increase at 1 h, reached at maximal level at 6 and 12 h, and returned to the control level by 24 h after i.c.v. administration with KA. In addition, KA-induced hippocampal CA3 neuronal death in C57BL6 (wild type) group was more pronounced compared to KA-induced hippocampal CA3 pyramidal cell death in α-CGRP knock-out (KO) group. Furthermore, sumatriptan, a CGRP releasing inhibitor, significantly protected the pyramidal cell death in CA3 hippocampal region induced by KA administered i.c.v. in ICR mice. Our results suggest that α-CGRP may play an important role in the regulation of KA-induced pyramidal cell death in CA3 region of the hippocampus.     

Effects of in vivo administration of kainic acid on the extracellular amino acid pool in the rabbit hippocampus

The effect of local administration of kainic acid in the rabbit hippocampus was studied; the hippocampus was perfused continuously in the freely moving animal with an implanted 0.3-mm dialysis fiber. The pattern of endogenous amino acids in the perfusate, reflecting extracellular amino acids, was monitored with liquid chromatography separation and fluorimetric detection of amino acid derivatives. Kainic acid was included in the perfusion medium for up to 70 min at 0.1-1.0 mM and, with time, induced epileptiform activity. Endogenous glutamic acid, taurine, and phosphoethanolamine levels were increased selectively at the lower perfusion concentrations of kainic acid. Long perfusion periods with higher concentrations increased the levels of virtually all amino acids. Perfusion of the hippocampus with depolarizing concentrations of potassium gave an amino acid response partly similar to that seen with kainic acid treatment. However, one notable difference between the two responses was that the extracellular concentration of glutamine, although not influenced by kainic acid, was significantly decreased after high potassium concentrations. These results confirm previous notions that kainic acid has a primarily excitatory effect, one manifestation of this effect being the release of glutamic acid.     

甲基苯丙胺中毒大鼠相关脑区多巴胺能神经毒性研究

目的：探讨MA中毒多巴胺能神经毒性的损伤机制。方法：将Wistar大鼠40只,随机分成对照组10只和实验组30只（实验组分成三个亚组,分为末次给药后1天组、4天组和7天组,n=10）。实验组给予20mg/kg的MA腹腔注射,对照组给予同样剂量的生理盐水,每天注射一次,注射时间为20：00,连续注射4天。分别于末次给药后1天,7天,14天处死实验大鼠,用免疫组织化学染色法（S-P法）和荧光分光光度计法检测大鼠中脑黑质致密区（SNC）、中脑腹侧被盖区（VTA）、前额叶皮质（PFC）以及纹状体（CPu）四个脑区的多巴胺神经元细胞的形态和数量的变化,对神经纤维进行灰度值分析。结果：1、黑质致密区和腹侧被盖区TH阳性细胞图像分析结果与细胞计数分析结果一致：与对照组相比,各实验组TH免疫反应阳性降低,差异具显著性（P〈0.05）,d1组开始降低（P〈0.05）,d7组达到低谷（P〈0.01）,d14天组黑质致密区和腹侧被盖区TH免疫反应阳性有不同程度的恢复（P〈0.05）。2、纹状体和前额叶皮质TH阳性纤维图像定量分析结果：各实验组TH免疫反应阳性均减低（P〈0.05）,d7组阳性反应最弱（P〈0.01）,d14组仍未恢复（P〈0.05）。3、黑质致密区、腹侧被盖区、纹状体及前额叶皮质荧光分光光度计检测DA递质含量结果：与上述免疫组化结果基本一致。结论：1、大鼠各脑区TH阳性表达和DA含量,均出现不同程度的减低。2、MA中毒大鼠各脑区DA递质含量的变化与TH的变化结果基本一致。     

Alterations of taurine in the brain of chronic kainic acid epilepsy model

Summary. The aim of the study was to investigate the changes of taurine in the kainic acid (KA, 10 mg/kg, s.c.) chronic model of epilepsy, six months after KA application. The KA-rats used were divided into a group of animals showing weak behavioural response to KA (WDS, rare focal convulsion; rating scale <2 up to 3 h after KA injection) and a group of strong response to KA (WDS, seizures; rating >3 up to 3 h after KA injection). The brain regions investigated were caudate nucleus, substantia nigra, septum, hippocampus, amygdala/piriform cortex, and frontal, parietal, temporal and occipital cortices. KA-rats with rating <2 developed spontaneous WDS which occurred chronically and six months after KA injection increased taurine levels were found in the hippocampus (125.4% of control). KA-rats with rating >3 developed spontaneous recurrent seizures and six months after injection increased taurine levels were found in the caudate nucleus (162.5% of control) and hippocampus (126.6% of control), while reduced taurine levels were seen in the septum (78.2% of control). In summary, increased taurine levels in the hippocampus may involve processes for membrane stabilisation, thus favouring recovery after neuronal hyperactivity. The increased taurine levels in the caudate nucleus could be involved in the modulation of spontaneous recurrent seizure activity.     

甲基苯丙胺中毒大鼠相关脑区多巴胺能神经毒性研究

目的:探讨MA中毒多巴胺能神经毒性的损伤机制。方法:将Wistar大鼠40只,随机分成对照组10只和实验组30只(实验组分成三个亚组,分为末次给药后1天组、4天组和7天组,n=10)。实验组给予20mg/kg的MA腹腔注射,对照组给予同样剂量的生理盐水,每天注射一次,注射时间为20:00,连续注射4天。分别于末次给药后1天,7天,14天处死实验大鼠,用免疫组织化学染色法(S-P法)和荧光分光光度计法检测大鼠中脑黑质致密区(SNC)、中脑腹侧被盖区(VTA)、前额叶皮质(PFC)以及纹状体(CPu)四个脑区的多巴胺神经元细胞的形态和数量的变化,对神经纤维进行灰度值分析。结果:1、黑质致密区和腹侧被盖区TH阳性细胞图像分析结果与细胞计数分析结果一致:与对照组相比,各实验组TH免疫反应阳性降低,差异具显著性(P<0.05),d1组开始降低(P<0.05),d7组达到低谷(P<0.01),d14天组黑质致密区和腹侧被盖区TH免疫反应阳性有不同程度的恢复(P<0.05)。2、纹状体和前额叶皮质TH阳性纤维图像定量分析结果:各实验组TH免疫反应阳性均减低(P<0.05),d7组阳性反应最弱(P<0.01),d14组仍未恢复(P<0.05)。3、黑质致密区、腹侧被盖区、纹状体及前额叶皮质荧光分光光度计检测DA递质含量结果:与上述免疫组化结果基本一致。结论:1、大鼠各脑区TH阳性表达和DA含量,均出现不同程度的减低。2、MA中毒大鼠各脑区DA递质含量的变化与TH的变化结果基本一致。     

Neuroexcitatory amino acids: phosphonic analogue of kainic acid

Summary The enantioselective synthesis of phosphonic analogue of kainic acid is described.     

Formation of the base modification 8-hydroxyl-2'-deoxyguanosine and DNA fragmentation following seizures induced by systemic kainic acid in the rat

The formation of oxidative DNA damage as a consequence of seizures remains little explored. We therefore investigated the regional and temporal profile of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) formation, a hallmark of oxidative DNA damage and DNA fragmentation in rat brain following seizures induced by systemic kainic acid (KA). Formation of 8-OHdG was determined via HPLC with electrochemical detection, and single- and double-stranded DNA breaks were detected using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated nick end-labeling (TUNEL), respectively. Systemic KA (11 mg/kg) significantly increased levels of 8-OHdG within the thalamus after 2 h, within the amygdala/piriform cortex after 4 h, and within the hippocampus after 8 h. Levels remained elevated up to sevenfold within these areas for 72 h. Smaller increases in 8-OHdG levels were also detected within the parietal cortex and striatum. PANT-positive cells were detected within the thalamus, amygdala/piriform cortex, and hippocampus 24-72 h following KA injection. TUNEL-positive cells appeared within the same brain regions and over a similar time course (24-72 h) but were generally lower in number. The present data suggest oxidative damage to DNA may be an early consequence of epileptic seizures and a possible initiation event in the progression of seizure-induced injury to DNA fragmentation and cell death.     

Estrogens influence behavioral responses in a kainic acid model of neurotoxicity

The behavioral and neuroprotective effects of 17beta-estradiol (E2), on ovariectomized rats treated with a subconvulsive dose (7 mg/kg bw, ip) of kainic acid (KA), were examined. Estradiol was administered either acutely (150 mug/rat, ip) along with KA, 14 days post-ovariectomy, or chronically (sc capsules providing proestrus estrogen levels in serum) starting at ovariectomy. Exploratory behavior, as deduced by sniffing in the open field test, was reduced in KA-treated rats. Both hormonal schemes partially restored sniffing behavior in KA-lesioned subjects. Moreover, acute and chronic E2 administration in KA-treated rats resulted in increased vertical and horizontal activity of these animals in the open field test. Memory for object recognition was reduced following KA and was not restored by hormonal treatments. Acute, but not chronic, E2 coadministration with KA significantly impaired spatial performance in the water maze task, while KA alone had no effect. Both acute and chronic estradiol administration rescued hilar and CA1 neurons from KA-induced cell death. Chronic, but not acute, E2 increased neurofilament immunoreactivity in the mossy fibers of the dentate gyrus neurons, similarly to KA. Our results show that although estradiol administration in KA-treated rats has beneficial effects on cell survival, it has diverse effects on exploratory behavior, object, and spatial memory. Estradiol effects on KA-lesioned animals depended on the duration and timing of exposure to the hormone, implying different mechanisms of hormone actions.     

Methamphetamine and lipid peroxidation in the rat retina

BACKGROUND: The use of psychoactive drugs during adolescence and early adult life has increased in the last few decades. It is known that developmental exposure to psychostimulants affects the sensory systems, and the retina has been shown to be a target tissue. This work was conducted to evaluate the pattern of lipid peroxidation in the rat retina following prenatal exposure to methamphetamine (MA). METHODS: Pregnant female Wistar rats were given MA (5 mg/kg of body weight/day; SC, in 0.9% saline) from GD 8 to 22. Offspring were sacrificed at postnatal days (PNDs) 7, 14, and 21. The retinas were homogenized, and both the total antioxidant and superoxide dismutase (SOD) activities were measured by enzymatic-colorimetric methods. The lipid peroxidation byproducts (malondialdehyde [MDA] and MDA-like metabolites) were measured by the thiobarbituric acid test. RESULTS: Total antioxidant levels were lower in the MA group at PND 21 in both males and females. The activity of SOD was higher in PND 7 females from the MA group. MDA levels were higher in the MA group at PND 21 in both genders. CONCLUSIONS: These findings suggest that prenatal-induced MA toxicity in the retina may be related to lipid peroxidation processes and oxidative stress.     

Effect of α-MSH upon cyclic AMP levels induced by the glutamatergic agonists NMDA, quisqualic acid, and kainic acid

This study was carried out to investigate possible interactions between some glutamatergic agonists and the peptide α-MSH upon the cyclic AMP levels. We used an in vitro tissue slice preparation incubated in the presence of different glutamatergic agonists such as N-methyl- -aspartic acid (NMDA), quisqualic acid (QUIS), kainic acid (KA), and the peptide α-MSH together with each agonist. Slices containing caudate putamen and accumbens were chosen according to neurochemical data indicating that the striatum contains a moderate amount of MSH binding sites and also receives glutamatergic innervation. Exposure of these slices to either MSH or to the agonists NMDA or QUIS resulted in an increase in the cAMP levels in relation to controls. Nevertheless, incubation with KA resulted in no changes in the nucleotide levels. The combination of MSH/NMDA induced a reduction of cAMP levels in relation to those obtained with NMDA alone. The combinations of QUIS/MSH or KA/MSH also induced variations in the values of nucleotide in relation to the those obtained with the peptide alone or with the corresponding agonist; these changes were related to the dose of agonist used in each case. The results obtained in these experiments suggest the existence of some interaction between the peptide and the agonist used.     

Transmitter release in hippocampal slices from rats with limbic seizures produced by systemic administration of kainic acid

The systemic injection of kainic acid (KA) has been shown to destroy neurons in the hippocampus and to induce limbic-type seizure activity. However, little is known on the neurochemical events that are associated with this convulsant effect. In the present work we studied the spontaneous and the K⁺-stimulated release of labeled -aminobutyric acid (GABA), glutamate, serotonin and dopamine, in hippocampal slices of KA-treated rats, at the moment of clinical seizures (2 h) and 72 h later. At the onset of convulsions we found a 40–45% decrease in the K⁺-stimulated release of GABA. The release of the other neurotransmitters was not significantly affected by KA treatment. After 72 h GABA release was still reduced by 30–40%. It is concluded that the epileptogenic effect of KA in the hippocampus is probably related to a diminished inhibitory GABAergic neurotransmission.     

Long-term kainic acid exposure reveals compartmentation of glutamate and glutamine metabolism in cultured cerebellar neurons

Glutamate neurotoxicity is implicated in most neurodegenerative diseases, and in the present study the long-term effects of the glutamate agonist kainic acid (KA) on cerebellar neurons are investigated. Primary cell cultures, mainly consisting of glutamatergic granule neurons, were cultured in medium containing 0.05 or 0.50 mM KA for 7 days and subsequently incubated in medium containing [U-¹³C]glutamate or [U-¹³C]glutamine. The amount of protein and number of cells were greatly reduced in cultures exposed to 0.50 mM KA compared to those exposed to 0.05 mM KA. Glutamine consumption was not affected by KA concentration, whereas that of glutamate was decreased by high KA, confirming reduction in glutamate transport reported earlier. Neurons cultured with 0.50 mM KA and incubated with glutamate contained decreased amounts of glutamate, aspartate and GABA compared to those cultured with 0.05 mM KA. Incubation of cells exposed to 0.50 mM KA with glutamine led to an increased amount of glutamate compared to cells exposed to 0.05 mM KA, whereas the intracellular amounts of aspartate and GABA remained unaffected by KA concentration. Furthermore, mitochondrial metabolism of -[U-¹³C]ketoglutarate derived from [U-¹³C]glutamate and [U-¹³C]glutamine was significantly reduced by 0.50 mM KA. The results presented illustrate differential vulnerability to KA and can only be understood in terms of inter- and intracellular compartmentation.     

牛蛙视网膜诱导型一氧化氮合酶免疫组化定位

用免疫组织化学方法研究了诱导型一氧化氮酶（iNOS)在牛蛙视网膜中的表达。结果显示,在正常状态视网膜中,无长突细胞呈弱阳性反应;节细胞层、双极细胞,水平细胞和光感受器内段呈阴性反应,在暗适应状态下,神经节细胞,内核层的无长突细胞呈强阳性反应;一些双极细胞,水平细胞和光感受器内段呈弱阳性反应,提示NO主要在暗适应状态下参与视网膜的信息传递过程。     

Prevention of kainic acid-induced changes in nitric oxide level and neuronal cell damage in the rat hippocampus by manganese complexes of curcumin and diacetylcurcumin

Curcumin is a natural antioxidant isolated from the medicinal plant Curcuma longa Linn. We previously reported that manganese complexes of curcumin (Cp-Mn) and diacetylcurcumin (DiAc-Cp-Mn) exhibited potent superoxide dismutase (SOD)-like activity in an in vitro assay. Nitric oxide (NO) is a free radial playing a multifaceted role in the brain and its excessive production is known to induce neurotoxicity. Here, we examined the in vivo effect of Cp-Mn and DiAc-Cp-Mn on NO levels enhanced by kainic acid (KA) and L-arginine (L-Arg) in the hippocampi of awake rats using a microdialysis technique. Injection of KA (10 mg/kg, i.p.) and L-Arg (1000 mg/kg, i.p.) significantly increased the concentration of NO and Cp-Mn and DiAc-Cp-Mn (50 mg/kg, i.p.) significantly reversed the effects of KA and L-Arg without affecting the basal NO concentration. Following KA-induced seizures, severe neuronal cell damage was observed in the CA1 and CA3 subfields of hippocampal 3 days after KA administration. Pretreatment with Cp-Mn and DiAc-Cp-Mn (50 mg/kg, i.p.) significantly attenuated KA-induced neuronal cell death in both CA1 and CA3 regions of rat hippocampus compared with vehicle control, and Cp-Mn and DiAc-Cp-Mn showed more potent neuroprotective effect than their parent compounds, curcumin and diacetylcurcumin. These results suggest that Cp-Mn and DiAc-Cp-Mn protect against KA-induced neuronal cell death by suppression of KA-induced increase in NO levels probably by their NO scavenging activity and antioxidative activity. Cp-Mn and DiAc-Cp-Mn have an advantage to be neuroprotective agents in the treatment of acute brain pathologies associated with NO-induced neurotoxicity and oxidative stress-induced neuronal damage such as epilepsy, stroke and traumatic brain injury.     

Histotypic organization of the rat retina in vitro

Summary Retinae from two- and three-day-old rats were explanted in plasma clots and grown in vitro with the flying coverslip method. After seven to seventeen days in culture, the retinal tissue was fixed with aldehydes and osmium tetroxide and embedded for examination with the electron microscope. Study of cross sections (perpendicular to the coverslip) revealed a histotypic pattern of organization, especially in the thicker regions of the explants. Layering of cells quite similar to that in the intact retina was seen to develop from the relatively primitive, explanted retinal epithelium. However, each layer contained fewer cells than its counterpart in vivo. All major neuronal cell types were distinguished by their location and cytological characteristics. Development of the saccules of sensory cell outer segments was observed to occur in vitro by an infolding of the plasma membrane. Synaptic ribbon complexes developed to the mature form in the outer plexiform layers, while conventional synapses were numerous in the inner plexiform layers. Synaptic ribbons were also seen in bipolar cell axons in the inner plexiform layers. Amacrine and ganglion cells in these regions were relatively sparse. A survey of posterior regions of noncultured three-day-old rat retinae showed no synapses of any sort; therefore the synapses in the cultures formed in vitro. The retina is recommended for studies of synaptogenesis in tissue culture, for it offers several advantages over expiants from other areas of the neuraxis, including a clear layering pattern, many identifiable cell processes with characteristic synaptic relationships between them, and a well-defined sequence of developmental events.Dedicated to Professor Wolfgang Bargmann on the occasion of his 65th birthday.     

Histological study of ibotenic acid-induced modifications of rat retina and their attenuation by diazepam

Summary After 2 h intraocular injections of 19 and 190 nmoles ibotenic acid in the rat retina produced an intensive vacuolization of the inner plexiform layer and cellular alterations, in the inner nuclear layer and ganglion cell layer. These alterations consisted of either cytoplasmic swelling accompanied by clumping of the nuclear chromatin or darkening of the cytoplasm along with nuclear condensation. A week later the retinas were thinner than the controls due to the disappearance of the affected cells. Pre-treatment with diazepam prevents the morphological alterations induced by 19 nmoles ibotenic acid; mainly the swelling, which was completely prevented, while the darkening was reduced drastically, although some vacuolization of the inner plexiform layer is still present.