Solubilization of Kainic Acid Binding Sites from Rat Brain

Kainic acid binding sites were solubilized from rat brain using a combination of Triton X-100 and digitonin. The highest percentage of solubilized binding sites (45%) was obtained by treating brain membranes with 1% Triton-X-100 and 0.2% digitonin in 0.5 M potassium phosphate containing 20% glycerol. The solubilized binding sites were stable and amenable to analysis by gel filtration and lectin affinity chromatography. Computer assisted analyses demonstrated that the solubilized sites displayed high- and low-affinity binding constants similar to the membrane-bound sites. Competition experiments further supported the pharmacological similarities of the solubilized and membrane-bound sites. Gel filtration chromatography of the solubilized binding site indicated that the detergent-bound complex had a Stokes radius of 82.7 A. The [3H]kainic acid binding site appears to be glycosylated based on its capability to bind to lectins. The lectin, wheatgerm agglutinin, proved to be a potentially useful tool for characterization because the solubilized binding sites were bound and eluted in relatively high yield.     

Changes in Regional Neurotransmitter Amino Acid Levels in Rat Brain During Seizures Induced by l-Allylglycine, Bicuculline, and Kainic Acid

Changes in amino acid concentrations were studied in the cortex, cerebellum, and hippocampus of the rat brain, after 20 min of seizure activity induced by kainic acid, 47 mumol/kg i.v.; L-allylglycine, 2.4 mmol/kg i.v.; or bicuculline, 3.27 mumol/kg i.v. in paralysed, mechanically ventilated animals. Metabolic changes associated with kainic acid seizures predominate in the hippocampus, where there are decreases in aspartate (-26%), glutamate (-45%), taurine (-20%), and glutamine (-32%) concentrations and an increase in gamma-aminobutyric acid (GABA) concentration (+ 26%). L-Allylglycine seizures are associated with generalized decreases in GABA concentrations (-32 to -54%), increases in glutamine concentrations (+10 to +53%), and a decrease in cortical aspartate concentration (-14%). Bicuculline seizures, in fasted rats, are associated with marked increases in the levels of hippocampal GABA (+106%) and taurine (+40%). In the cerebellum, there are increases in glutamine (+50%) and taurine concentrations (+36%). These changes can be explained partially in terms of known biochemical and neurophysiological mechanisms, but uncertainties remain, particularly concerning the cerebellar changes and the effects of kainic acid on dicarboxylic amino acid metabolism.     

Ontogeny of Receptor Binding Sites for [3H]Glutamic Acid and [3H]Kainic Acid in the Rat Cerebellum

The development of the specific binding sites for L-[3H]glutamic acid (KD = 370 nM) and for [3H]kainic acid (KD = 39 nM) was studied in the rat cerebellum. Specific binding at both sites remains low during the first week after birth but increases markedly during the second and third weeks after birth, when glutamatergic parallel fiber synaptogenesis occurs. The development of the kainate site lags behind that of the glutamate site, indicating their autonomy.     

雷公藤甲素对KA损伤大鼠星形胶质细胞的影响

目的:观察海人藻酸(Kainic acid,KA)海马内注射后星形胶质细胞的变化及雷公藤甲素(TRP)对其的影响。方法:90只SD大鼠(200～220g)随机分为3组:右侧海马注射生理盐水后生理盐水灌胃作为对照组(NS NS),右侧海马注射海人藻酸后生理盐水灌胃干预组(KA NS),右侧海马注射海人藻酸后雷公藤甲素灌胃干预组(KA TRP)。动物存活1天,3天,5天,7天,14天后免疫组织化学结合图像分析技术观察海马内星形胶质细胞形态和数目的变化。结果:(KA NS)组海马内星形胶质细胞数目明显增多,胞体明显增大,突起变短,变粗,与(NS NS)组相比差别具有显著性(p<0.05);(KA TRP)组星形胶质细胞数量明显减少,胞体变小,突起变细长,与(KA NS)组相比差别具有显著性(P<0.05)。结论:KA注射后可导致大鼠海马内星形胶质细胞的激活,雷公藤甲素对KA诱导的星形胶质细胞的活化有抑制作用。     

Amygdala Kindling Modifies Extracellular Opioid Peptide Content in Rat Hippocampus Measured by Microdialysis

Abstract: Opioid peptide release in the hippocampus was shown to be increased immediately following amygdala kindling stimulation in freely moving rats using microdialysis combined with a universal opioid peptide radioimmunoassay (RIA). Extracellular opioid peptide levels were elevated (55% above basal levels) within the first 10 min after electrical stimulation-induced partial seizures in previously nonkindled animals. Fully kindled rats showed lower extracellular opioid peptide levels (40% reduction) during the interictal period [16 ± 2.1 days (mean ± SEM) after the last stage V seizure], in comparison with values obtained from the sham-kindled group under basal conditions. However, opioid peptide release in fully kindled rats increased above 152% of interictal levels within the first 20 min after onset of fully kindled seizures, attaining peak levels equal to that of the partial kindled group and returning to prestimulation conditions 40–60 min following the ictal events. The majority of the immunoreactive material recovered from the hippocampus within the first 20 min following partial and generalized kindled seizures coeluted with dynorphin-A (1–6), dynorphin-A (1–8), and Leu-enkephalin by HPLC/RIA analysis. It is proposed that the enhanced opioid peptide release in hippocampus induced by amygdala kindling stimulation might be associated with either enhanced excitability or seizure suppression as seizure susceptibility fluctuates. The reduced interictal opioid peptide levels may also underlie some interictal behavioral disturbances.     

Role of Guanine Nucleotides as Endogenous Ligands of a Kainic Acid Binding Site Population in the Mammalian Cerebellum

Abstract: An endogenous inhibitor of the membrane binding of kainic acid was extracted from pig brain tissue and purified. The substance was identified as GMP by structural analysis: Most likely it corresponds to an inhibitor previously extracted from the rat brain. The nucleotide is active as an inhibitor for kainate binding on goldfish brain synaptosomes, probably owing to direct displacement on receptor sites; it is also active on a low-affinity kainate site population in membranes from rat cerebellum. The interaction of GMP with the latter sites leads to a concentration-dependent kainate binding increase or inhibition, thus demonstrating that these sites can bind the nucleotide and cooperatively increase their affinity. Other guanine nucleotides show interaction with these sites, by either an increase (GTP) or inhibition (cyclic GMP or GDP) of kainate binding. These findings support the view that a guanine nucleotide is the endogenous ligand of a receptor in the mammalian cerebellum similar to the kainate binding protein present with high density in the cerebellum of lower vertebrates, whose function is probably connected to the role of the glial cells in this zone.     

Resveratrol Protects Against Neurotoxicity Induced by Kainic Acid

Increased oxidative stress has been implicated in the mechanisms of excitotoxicity in hippocampus induced by kainic acid (KA), an excitatory glutamate receptor agonist. Resveratrol, a polyphenolic antioxidant compound enriched in grape, is regarded as an important ingredient in red wine to offer cardiovascular and neural protective effects. This study was designed to investigate whether resveratrol treatment may ameliorate neuronal death after KA administration. Adult Sprague Dawley male rats were treated with KA (8 mg/kg) daily for 5 days and another group was treated similarly with KA plus resveratrol (30 mg/kg/day). Three hr after the last treatment protocol, animals were sacrificed, and brain sections were obtained for histochemical and immunohistochemical identification of neurons, astrocytes and microglial cells. After KA administration, significant neuronal death and activation of astrocytes and microglial cells were observed in the hippocampal CA1, CA3 and polymorphic layer (hilar) of the dentate gyrus (DG) (P < 0.001). The KA-induced hippocampal neuronal damage was significantly attenuated by treatment with resveratrol (P < 0.001). Resveratrol also suppressed KA-induced activation of astrocytes and microglial cells. Since increased oxidative stress is a key factor for KA-induced neurotoxicity, this study demonstrated the ability of resveratrol to act as free radical scavenger to protect against neuronal damage caused by excitotoxic insults.Special issue dedicated to Dr. Lawrence F. Eng.     

Presence of Two Benzodiazepine Binding Sites in the Rat Hippocampus

Abstract: Characteristics of receptor binding of diazepam and flunitrazepam in three brain areas were compared. It was found that in the cerebral cortex and cerebellum the number of sites was similar for both ligands and that the affinity of diazepam was four times lower than the affinity of flunitrazepam. In contrast, when binding in the hippocampus was analyzed (assuming the presence of homogenous binding sites), it was found that the number of binding sites was higher and that the affinity was 17 times lower for diazepam than for flunitrazepam. This difference is due to the presence of two diazepam binding sites in this brain area, as demonstrated by a Scatchard analysis.     

Thyrotropin-Releasing Hormone Receptor Binding Sites: Autoradiographic Distribution in the Rat and Guinea Pig Brain

Thyrotropin-releasing hormone (TRH) binding sites were labeled in vitro in mounted brain tissue sections from rat and guinea pig brains with [3H]methyl TRH and localized autoradiographically using 3H-sensitive film. Regional densities of TRH binding sites were measured by computer-assisted microdensitometry. The distribution of sites in both species was highly heterogeneous. In both guinea pig and rat brains, the highest densities of binding sites were seen in the amygdaloid nuclei and the perirhinal cortex. In contrast, in other brain areas, a clear difference between the distribution of sites in rat and guinea pig was found. The temporal cortex, pontine nuclei, and interpeduncular nucleus, which contained high densities of binding in the guinea pig, were scarcely labeled in the rat. The accessory olfactory bulb and the septohippocampal area presented in the rat higher concentrations of binding sites than in the guinea pig. Other brain areas showing intermediate to low densities in both species were accumbens nucleus, bed nucleus of the stria terminalis, dentate gyrus, facial and hypoglossal nuclei, and gelatinosus subnucleus of the trigeminal nerve, among others. The anterior pituitary also presented low to intermediate concentrations of receptors. The distribution of TRH sites here described does not completely correlate with that of endogenous TRH, but is in good agreement with previous biochemical data. The results are discussed in correlation to the physiological effects that appear to be mediated by TRH.     

γ-Aminobutyric Acid Turnover in Rat Striatum: Effects of Glutamate and Kainic Acid

The turnover rate of gamma-aminobutyric acid (GABA) in the rat striatum was estimated by measuring its accumulation after inhibition of GABA-transaminase (GABA-T) with gabaculine. Intrastriatal injections of 100 micrograms gabaculine induced a rapid and complete inhibition of GABA-T. GABA accumulation was linear with time for at least 60 min (estimated turnover rate = 25 nmol/mg protein/h). The accumulation of GABA after gabaculine administration in animals that had been treated with kainic acid (5 nmol intrastriatally, 7 days) was only 40% of the control value, indicating that a major fraction of the net increase in GABA content induced by gabaculine originates in kainic acid-sensitive neurons. Intrastriatal injection of a mixture of kainic acid (5 nmol) and gabaculine caused a net increase in striatal GABA content significantly greater than that observed in controls, suggesting that neuronal death induced by kainic acid is preceded by a period of increased neuronal activity. Glutamic acid, the putative neurotransmitter for the excitatory corticostriatal pathway, also produced a significant increase in striatal GABA accumulation when injected together with gabaculine. This effect was blocked by the administration of the glutamate receptor antagonist glutamic acid diethyl ester. The interactions between GABAergic neurons and other neurotransmitters present in the striatum were also analyzed.     

γ-Aminobutyric Acid and Benzodiazepine Binding Sites in Audiogenic Seizure-Susceptible Mice

Abstract: The properties of γ-aminobutyric acid recognition sites, benzodiazepine binding sites and the effect of exogeneous γ-aminobutyric acid on benzodiazepine binding were determined in crude membrane fractions prepared from the brains of DBN/2 mice at ages before (8-9 and 17-18 days), during (22-23 and 28-29 days) and after (40-43 days) the age of high susceptibility to audiogenic seizures. These have been compared with data from age- matched mice of a strain (TO) with lower audiogenic seizure susceptibility. The number of high-affinity [³H]γ-aminobutyric acid binding sites was lower at all ages in DBN/2 mice compared with TO mice, but the affinity was higher in DBN/2 mice. The number of low-affinity [³H]y-aminobutyric acid binding sites was lower at 8-9 days and 40-43 days in DBN/2 mice, but was not significantly different from TO mice at other ages. For [³H]flunitrazepam binding, the only difference found was a slight reduction in the number of binding sites at 28-29 days of age in DBN/2 mice. γ-Aminobutyric acid stimulation of [³H]-flunitrazepam binding was not significantly different up to 22-23 days of age, but was higher in DBN/2 mice at 28-29 days and lower at 40-43 days. Impairment of γ-aminobutyric acid function is a possible permissive factor in the age-dependent audiogenic seizure susceptibility in DBN/2 mice.     

Coupling of Inositol Phospholipid Metabolism with Excitatory Amino Acid Recognition Sites in Rat Hippocampus

Ibotenate, a rigid structural analogue of glutamate, markedly enhances the hydrolysis of membrane inositol phospholipids, as reflected by the stimulation of [3H]inositol monophosphate formation in rat hippocampal slices prelabeled with [3H]inositol and treated with Li+. Quisqualate, homocysteate, L-glutamate, and L-aspartate also induce a significant (albeit weaker) increase in [3H]inositol monophosphate formation, whereas N-methyl-D-aspartate, kainate, quinolinate, and N-acetylaspartylglutamate are inactive. The increase in [3H]inositol monophosphate formation elicited by the above-mentioned excitatory amino acids is potently and selectively antagonized by DL-2-amino-4-phosphonobutyric acid, a dicarboxylic amino acid receptor antagonist. These results suggest that, in the hippocampus, a class of dicarboxylic amino acid recognition sites is coupled with phospholipase C, the enzyme that catalyzes the hydrolysis of membrane inositol phospholipids.     

Effects of Kainic Acid in Rat Brain Synaptosomes: The Involvement of Calcium

Abstract: The effects of kainic acid were investigated in preparations of rat brain synaptosomes. It was found that kainic acid inhibited competitively the uptake of d -[³H]aspartate, with a K _i of approximately 0.3 m m . Kainic acid also caused release of two excitatory amino acid neurotranstnitters, aspartate and glutamate, in a time- and concentration-dependent manner, but had no effect on the content of γ-aminobutyric acid. Concomitant with the release of aspartate and glutamate, depolarization of the synaptosomal membrane and an increase in intracellular calcium were observed, with no measurable change in the concentration of internal sodium ions. The increase in intrasynaptosomal calcium and decrease in transmem-brane electrical potential were prevented by the addition of glutamate, whereas the kainate-induced release of ra-dioactive aspartate was substantially inhibited by lowering the concentration of calcium in the external medium. It is postulated that kainic acid reacts with a class of glutamate receptors located in a subpopulation of synaptosomes, presumably derived from the glutamatergic and aspartatergic neuronal pathways, which possesses high-affinity uptake system(s) for glutamate and/or aspartate. Activation of these receptors causes opening of calcium channels, influx of calcium into the synaptosomes, and depolarization of the synaptosomal plasma membrane with consequent release of amino acid neurotransmitters.     

Decreased Expression of Calmodulin Kinase II and Calcineurin Messenger RNAs in the Mouse Hippocampus After Kainic Acid-Induced Seizures

Abstract: The Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and the phosphatase calcineurin (CaN) are especially abundant in the mammalian CNS, where they have been implicated repeatedly in different neuronal functions. CaMKII is a holoenzyme that is likely to be constituted of both homomultimers and heteromultimers, CaMKIIα and CaMKIIβ being the most abundant subunits in the brain. CaN is a heterodimer constituted of a catalytic subunit (CaN A) and a regulatory subunit (CaN B), and CaN Aα is the predominant form in the brain. We studied the expression of CaMKIIα, CaMKIIβ, and CaN Aα subunit messenger RNAs in the mouse hippocampus at different times after the administration of a convulsant dose of kainic acid. CaMKIIα and CaN A immunohistochemistry was also performed. We observed a transient decrease in the three messenger RNAs in the kainic acid-treated mice, peaking at 5 or 24 h of treatment. The effect had disappeared completely 8 days after treatment. No significant alterations in CaMKII or CaN immunolabelling were observed in the hippocampus of kainic acid-treated mice. The observed modifications could be due to the neuronal hyperexcitability induced by kainic acid rather than neuronal degeneration, because no areas of neuronal loss were detected. Our results suggest that the expression of CaMKII and CaN mRNAs is down-regulated in neuronal cells in response to the hyperexcitability induced by kainic acid. The transient nature of the effect and the apparent absence of significant modifications in the amount of their corresponding proteins may be related to the absence of neuronal damage.     

Somatostatin Precursor in the Rat Striatum: Changes After Local Injection of Kainic Acid

The molecular forms of somatostatin contained in the rat striatum were separated by size-exclusion HPLC. Three major peaks of somatostatin-like immunoreactivity (SLI) were resolved. Two peaks cochromatographed with synthetic somatostatin-14 (SS-14) and somatostatin-28 (SS-28), respectively. One peak exhibited a higher molecular weight (about 10,000) and may contain a proform of somatostatin. Local injection of the neurotoxin kainic acid (1 microgram) into the left striatum resulted in a persistent decrease (65-85%) of all three forms of somatostatin. In the contralateral--not injected--striatum a decrease of SLI was also observed which was maximal (45%) after 2 days and was largely abolished after 7 days. This decrease of SLI in the contralateral striatum, however, was due mainly to a decrease of SS-14 and SS-28 but not of the putative proform. Our data suggest that kainic acid causes a destruction of somatostatin-containing perikarya in the injected striatum, whereas in the contralateral striatum increased release with subsequent inactivation of SS-14 and SS-28 takes place. The putative somatostatin proform may serve as neurochemical marker for somatostatin-containing perikarya in the striatum.     

Inhibition of Axoplasmic Transport in the Optic System by Kainic Acid

Axoplasmic transport along the optic axons was studied after intraocular injections of kainic acid (KA). Transport of labeled material did not initiate from the eye when KA was injected simultaneously with the protein precursor [3H]proline. When KA was injected after axoplasmic transport of labeled proteins had begun, no additional radioactive material moved out of the retinal ganglion cells. However, the labeled material already present in the optic nerve at the time of KA injection continued to move, and accumulated at the nerve endings. Although KA reduces the incorporation of precursor, this effect of KA on axoplasmic transport appears to be more than a consequence of inhibition on precursor uptake or protein synthesis. Recovery from this KA action began 6 h after exposure to KA and was about 50% recovered by 36 h. The extent of the recovery remained at this level for as long as a week, which suggested a partial recovery of the ganglion cells. A second exposure to KA after the inner plexiform layer had virtually disappeared was as effective as the first exposure in preventing the appearance of transported protein in the optic nerve, suggesting a direct action of KA on the ganglion cells. We interpreted the results to indicate that KA interferes with the initiation phase of axoplasmic transport in ganglion cells and this effect is partially reversible.     

Glucocorticoids Exacerbate Kainic Acid–Induced Extracellular Accumulation of Excitatory Amino Acids in the Rat Hippocampus

Glucocorticoids (GCs) compromise the ability of hippocampal neurons to survive various insults, and do so, at least in part, by exacerbating steps in the glutamate/N-methyl-D-aspartate (NMDA)/calcium cascade of damage. As evidence, GCs impair uptake of glutamate by hippocampal astrocytes, the GC endangerment of the hippocampus is NMDA receptor dependent, and GCs exacerbate kainic acid (KA)-induced calcium mobilization. These observations predict that GCs should also exacerbate KA-induced accumulation of extracellular glutamate and aspartate. To test this, adrenalectomized rats were given replacement GCs in either the low or high physiological range. Three days later, rats were anesthetized and 1 mM KA was infused through a dialysis probe placed in the dorsal hippocampus. Extracellular amino acid concentrations in the dialysate were then assessed by HPLC. After KA infusion, high-GC rats (30 +/- 3 micrograms/dl) had significantly elevated concentrations of glutamate and aspartate compared with low-GC rats (all less than 0.95 micrograms/dl). The glutamate accumulation was due to GCs raising pre-KA concentrations, whereas the aspartate accumulation was due to GCs exacerbating the KA-induced rise. Glutamine concentrations were unaffected by KA, whereas the high-GC regimen elevated glutamine concentrations both before and after KA. Taurine concentrations rose after infusion of KA, but were unaffected by GC regime, whereas alanine concentrations were unaffected by either manipulation. Serine concentrations were unaffected by KA, but were depressed both before and after KA in high-GC rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration in Neuronal-Glial Metabolism of Glutamate by the Neurotoxin Kainic Acid

The effect of the excitotoxin kainic acid on glutamate and glutamine metabolism was studied in cerebellar slices incubated with D-[2-14C]glucose, [U-14C]gamma-aminobutyric acid, [3H]acetate, [U-14C]glutamate, and [U-14C]glutamine as precursors. Kainic acid (1 mM) strongly inhibited the labeling of glutamine relative to that of glutamate from all precursors except [2-14C]glucose and [U-14C]glutamine. Kainic acid did not inhibit glutamine synthetase directly. The data indicate that in the cerebellum kainic acid inhibits the synthesis of glutamine from the small pool of glutamate that is thought to be associated with glial cells. Kainic acid also markedly stimulated the efflux of glutamate from cerebellar slices and this release was not sensitive to tetrodotoxin. Kainic acid stimulated efflux of both glucose- and acetate-labeled glutamate. In contrast, veratridine released glucose-labeled glutamate preferentially via a tetrodotoxin-sensitive mechanism. Kainic acid did not release [U-14C]glutamate from synaptosomal fractions. These results suggest that the bulk of the glutamate released from cerebellar slices by kainic acid comes from nonsynaptic pools.     

Effects of Kainic Acid on High-Energy Metabolites in the Mouse Striatum

Abstract: Intrastriatal injection of either kainic acid (0.35 μg) or ibotenic acid (7.0 μg) in the mouse causes a profound and selective degeneration of striatal neurons accompanied by a secondary astrocytic response. The kainate injection (0.35 μg) resulted in significant decrements in the striatal levels of phosphocreatine and ATP by 30 min, a progressive reduction in adenosine phosphates between 30 min and 48 h, and a decrease in energy charge; whereas lactate levels increased by 44% at 2 h, glucose levels fell by 56%. Two hours after intrastriatal injection of ibotenic acid (7.0 μg) similar alternations in striatal high-energy phosphates and glucose disposition were found. Prior decortication protected against the neurotoxic effects of kainate in the mouse striatum and prevented the alterations in high-energy phosphates at 2 h although lactate levels increased by 212%. These findings in vivo are consistent with the hypothesis that the neurotoxic effects of acidic excitatory amino acids involve a profound activation of energy consumption by affected neurons.     

Characterization of Two [3H]Glutamate Binding Sites in Rat Hippocampal Membranes

The specific binding of L-[3H]glutamate was investigated in the presence and the absence of sodium ions in freshly prepared membranes from rat hippocampus. Sodium ions were found to have a biphasic effect; low concentrations induced a marked inhibition of the binding (in the range 0.5-5.0 mM), whereas higher concentrations resulted in a dose-dependent stimulation of binding (in the range 10-150 mM). These results permit the discrimination of two binding sites in hippocampal membranes. Both Na+-independent and Na+-dependent binding sites were saturable, exhibiting dissociation constants at 30 degrees C of 750 nM and 2.4 microM, respectively, with Hill coefficients not significantly different from unity, and maximal number of sites of 6.5 and 75 pmol/mg protein, respectively. [3H]Glutamate binding to both sites reached equilibrium between 5 and 10 min and was reversible. The relative potencies of a wide range of compounds, with known pharmacological activities, to inhibit [3H]glutamate binding were very different for the Na+-independent and Na+-dependent binding and suggested that the former sites were related to post-synaptic glutamate receptors, whereas the latter were related to high-affinity uptake sites. This conclusion was also supported by the considerable variation in the regional distribution of the Na+-dependent binding site, which paralleled that of the high-affinity glutamate uptake; the Na+-independent binding exhibited less regional variation.