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.     

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.     

不同年龄大鼠学习记忆能力及旷场行为比较

目的：实验对２２日龄、１月龄、５月龄、１０月龄、２４月龄的ＳＤ大鼠的行为学进行比较。方法：采用水迷宫及旷场分析法对上述五种不同年龄组的大鼠进行研究,数据采用多因素方差分析处理。结果：幼鼠（２２日龄、１月龄）及老年鼠（２４月龄）游泳所用时间及错误次数比成年鼠（５月龄、１０月龄）多,差异显著（Ｐ＜０．０５）。幼鼠及老年鼠的空间认知能力显著低于成年鼠,并且对新异环境的适应性差,更加紧张（Ｐ＜０．０５）。结论：动物的学习记忆能力,空间认知能力及兴奋性存在着年龄上的差异,青壮年大鼠学习记忆能力及对新异环境的适应性均强于幼年及老年大鼠     

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.     

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.     

Caspase 3 involves in neuroplasticity,microglial activation and neurogenesis in the mice hippocampus after intracerebral injection of kainic acid

Background

The roles of caspase 3 on the kainic acid-mediated neurodegeneration, dendritic plasticity alteration, neurogenesis, microglial activation and gliosis are not fully understood. Here, we investigate hippocampal changes using a mouse model that receive a single kainic acid-intracerebral ventricle injection. The effects of caspase 3 inhibition on these changes were detected during a period of 1 to 7 days post kainic acid injection.

Result

Neurodegeneration was assessed by Fluoro-Jade B staining and neuronal nuclei protein (NeuN) immunostaining. Neurogenesis, gliosis, neuritic plasticity alteration and caspase 3 activation were examined using immunohistochemistry. Dendritic plasticity, cleavvage-dependent activation of calcineurin A and glial fibrillary acidic protein cleavage were analyzed by immunoblotting. We found that kainic acid not only induced neurodegeneration but also arouse several caspase 3-mediated molecular and cellular changes including dendritic plasticity, neurogenesis, and gliosis. The acute caspase 3 activation occurred in pyramidal neurons as well as in hilar interneurons. The delayed caspase 3 activation occurred in astrocytes. The co-injection of caspase 3 inhibitor did not rescue kainic acid-mediated neurodegeneration but seriously and reversibly disturb the structural integrity of axon and dendrite. The kainic acid-induced events include microglia activation, the proliferation of radial glial cells, neurogenesis, and calcineurin A cleavage were significantly inhibited by the co-injection of caspase 3 inhibitor, suggesting the direct involvement of caspase 3 in these events. Alternatively, the kainic acid-mediated astrogliosis is not caspase 3-dependent, although caspase 3 cleavage of glial fibrillary acidic protein occurred.

Conclusions

Our results provide the first direct evidence of a causal role of caspase 3 activation in the cellular changes during kainic acid-mediated excitotoxicity. These findings may highlight novel pharmacological strategies to arrest disease progression and control seizures that are refractory to classical anticonvulsant treatment.     

Altered expression of sphingosine kinase 1 and sphingosine-1-phosphate receptor 1 in mouse hippocampus after kainic acid treatment

Kainic acid (KA) induces hippocampal cell death and astrocyte proliferation. There are reports that sphingosine kinase (SPHK)1 and sphingosine-1- phosphate (S1P) receptor 1 (S1P₁) signaling axis controls astrocyte proliferation. Here we examined the temporal changes of SPHK1/S1P₁ in mouse hippocampus during KA-induced hippocampal cell death. Mice were killed at 2, 6, 24, or 48 h after KA (30 mg/kg) injection. There was an increase in Fluoro-Jade B-positive cells in the hippocampus of KA-treated mice with temporal changes of glial fibrillary acidic protein (GFAP) expression. The lowest level of SPHK1 protein expression was found 2 h after KA treatment. Six hours after KA treatment, the expression of SPHK1 and S1P₁ proteins steadily increased in the hippocampus. In immunohistochemical analysis, SPHK1 and S1P₁ are more immunoreactive in astrocytes within the hippocampus of KA-treated mice than in hippocampus of control mice. These results indicate that SPHK1/S1P₁ signaling axis may play an important role in astrocytes proliferation during KA-induced excitotoxicity.     

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.     

Neuroexcitatory amino acids: phosphonic analogue of kainic acid

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

Bruce/apollon promotes hippocampal neuron survival and is downregulated by kainic acid

Prolonged or excess stimulation of excitatory amino acid receptors leads to seizures and the induction of excitotoxic nerve cell injury. Kainic acid acting on glutamate receptors produces degeneration of vulnerable neurons in parts of the hippocampus and amygdala, but the exact mechanisms are not fully understood. We have here investigated whether the anti-apoptotic protein Bruce is involved in kainic acid-induced neurodegeneration. In the rat hippocampus and cortex, Bruce was exclusively expressed by neurons. The levels of Bruce were rapidly downregulated by kainic acid in hippocampal neurons as shown both in vivo and in cell culture. Caspase-3 was activated in neurons exhibiting low levels of Bruce causing cell death. Likewise, downregulation of Bruce using antisense oligonucleotides decreased viability and enhanced the effect of kainic acid in the hippocampal neurons. The results show that Bruce is involved in neurodegeneration caused by kainic acid and the downregulation of the protein promotes neuronal death.     

A study on specific behavioral effects of formaldehyde in the rat

It has been reported that single exposure of rats of low-level formaldehyde vapor concentrations causes significant alteration in their motor activity in the inhalation chamber. In this study, we determined the effects of formaldehyde on the locomotor activity and behavior of adult male and female Lew. 1K rats in an open field two hours after termination of a single two hours lasting inhalative exposure to approximately 0.1, 0.5, or 5 ppm. Following behavioral parameters were quantitatively examined: numbers of crossed floor squares, occurrence frequencies of air and floor sniffing, grooming, rearing, and wall climbing, as well as the incidence of fecal boli. In the open field situation, the males of all formaldehyde groups crossed significantly lower numbers of floor squares. Furthermore, significant decrease in the occurrence frequencies of floor sniffing, rearing, and wall climbing were observed. Within the female rat groups exposed to 0.5 or 5 ppm formaldehyde, a significantly decreased numbers of crossed squares were registered, while this parameter remained unchanged in the 0.1 ppm group. Other parameters were also affected by the formaldehyde inhalation (e.g. significant increase in the occurrence frequencies of air sniffing in the 0.1 and 0.5 ppm groups and significant decrease in the numbers of floor sniffing in the 0.5 and 5 ppm groups, respectively). The incidence of fecal boli was not affected in any exposure group neither in males nor in females. It is concluded from the results obtained that formaldehyde significantly affects the locomotor behavior of adult male and female rats in the open field after a single inhalative exposure to the above mentioned concentrations.     

Methamphetamine exacerbates the toxic effect of kainic acid in the adult rat retina

The recreational use of the psychoactive drug, methamphetamine has increased markedly over the last three decades. It has long been known that this drug has detrimental effects upon the mammalian brain monoaminergic system, but the long- or short-term effects on the retina, a neurological extension of the central nervous system, have received little attention. The aim of this study was, therefore, to determine whether intraocular injection of methamphetamine (MA) is toxic to the healthy adult rat retina and to analyse its effects on the compromised retina after an injection of the ionotropic glutamate receptor agonist, kainate, which is known to cause retinal neuropathology. The equivalent of 1 mM (in the vitreous humour) MA and/or kainate (40 μM) were injected intravitreally. Flash electroretinograms (ERGs) were recorded before and 2 and 4 days after treatment. Five days after treatment, animals were killed and the retinas analysed either for the immunohistochemical localisation of various antigens or for electrophoresis/Western blotting. Some animals were kept for 19 days after treatment and the retinas analysed for tyrosine hydroxylase immunoreactivity. No differences could be found between vehicle- and MA-treated retinas with respect to the nature or localisation of either tyrosine hydroxylase immunoreactivity after 5 or 19 days or other antigens after 5 days. Moreover, the normal ERG and GFAP and calretinin protein antigens were unaffected by MA. Kainate treatment, however, caused a change in the ERGs after 2 and 4 days, an alteration in every antigen localised by immunohistochemistry and an increase in the retinal levels of calretinin and GFAP proteins. Significantly, the changes seen in the b-wave amplitude and implicit time of the ERG after 4 days and the increased level of GFAP protein after 5 days following kainate treatment were enhanced when MA was co-injected. Intravitreal injection of methamphetamine had no detectable detrimental effect on the normal adult rat retina but exacerbated the damaging effects of kainic acid. Such data suggest that a neurotoxic effect of MA may be more obviously illustrated when the tissue is already compromised as occurs in, for example, ischemia.     

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.     

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.     

Neuro-protective effects of carbamazepine on sleep patterns and head and body shakes in kainic acid-treated rats

The aim of this work was to analyze the effect of carbamazepine (CBZ) on sleep patterns and on “head and body shakes” and to determine the role of serotonin (5-HT) in a model of kainic-induced seizures. Thirty male Wistar rats (280–300 g) were used for polygraphic sleep recording. After a basal recording, the rats were allocated into three groups: kainic acid-treated animals (KA; 10 mg/kg; n = 10), carbamazepine-treated animals (CBZ; 30 mg/kg; n = 10) and animals injected with KA 30 min after pretreatment with CBZ (CBZ + KA; n = 10). Polygraphic recordings were performed for 10 h for 3 days, with the exception of the CBZ group, which were observed for 1 day. In order to measure the head and body shakes that occurred over that time, a behavioral assessment was performed in two additional groups of KA (n = 10) and CBZ + KA (n = 10) animals. After 10 h of behavioral assessment, the rats were sacrificed, and the levels of 5-HT and 5-hydroxy-indol-acetic acid (5-HIAA) were analyzed. We compared these findings with the results from a group of rats without pharmacological intervention (n = 10). All of the recordings were performed from 08:00 to 18:00 h. Data analysis: the electrographic parameters, head and body shake counting and monoamine concentrations were analyzed by an ANOVA test. Differences of *p ≤ 0.01 and **p ≤ 0.001 were considered statistically significant. Our results showed that CBZ exerted a protective effect on sleep pattern alterations induced by KA, which when administered alone caused a complete inhibition of sleep for the first 10 h after administration. Although there was a reduction in the amount of sleep after the administration of KA in CBZ-pretreated animals, sleep inhibition was incomplete. In addition, CBZ decreased the frequency of head and body shakes by 60% as compared to KA. The 5-HT and 5-HIAA levels in the pons were increased in the KA and KA + CBZ groups. Our conclusion is that in addition to decreasing seizure intensity, CBZ facilitates the partial recovery of sleep. These results suggest that CBZ provides neuro-protective effects on sleep and against seizures.     

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.     

Quinolinate-like neurotoxicity produced by aminooxyacetic acid in rat striatum

Summary The endogenous tryptophan metabolite quinolinic acid elicits in rodent brain a pattern of neuronal degeneration which resembles that caused by L-glutamate. Its qualities as a neurotoxic agent raised the hypothesis that quinolinic acid might be involved in the pathogenesis of human neurodegenerative disorders. Kynurenic acid, another endogenous tryptophan metabolite and preferential N-methyl-D-aspartate (NMDA) antagonist, has been shown to block quinolinic acid neurotoxicity. Here we report that microinjections of aminooxyacetic acid (AOAA), an inhibitor of kynurenine transaminase and of other pyridoxal phosphate-dependent enzymes, into the rat striatum produce neuronal damage resembling that caused by quinolinic acid. AOAA-induced striatal lesions can be prevented by kynurenic acid and the selective NMDA antagonist 2-amino-7-phosphonoheptanoic acid. These results suggest that AOAA produces excitotoxic lesions by depleting brain concentrations of kynurenic acid (inhibition of synthetic enzyme) or due to impairment of intracellular energy metabolism (depletion of cell energy resources). The concept of deficient neuroprotection due to metabolic defects might help to clarify the pathogenesis of human neurodegenerative disorders and to develop strategies that may be useful in their treatment.This work was supported by research grant from the Polish Academy of Sciences.These data have been communicated to the International Congress on Amino Acid Research held in Vienna in August 7–12, 1989.     

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.