Analysis of 3H-kainic acid binding to brain membranes in rats and frogs

It has been shown that H3-kainic acid (3H-KA) specifically binds with membrane preparations from various parts of rat brain or whole frog brain. The saturation isotherms of 3H-KA binding revealed the presence of two sites with a high and low affinity. An exception was for rat cerebellum where Scatchard analysis showed but one low affinity site. The density of 3H-KA binding sites in frog brain was 5 to 10 times higher than in rat brain. Among the drugs studied, KA itself, L-glutamate and folic acid were the most potent inhibitors of specific binding. Methyltetrahydrofolate, quinolinic acid, kynurenine, GABA, taurine, L-aspartate were ineffective in this respect. The kinetic analysis of the binding data in the presence or absence of L-glutamate and folic acid showed, however, that these drugs inhibited 3H-KA binding in a noncompetitive manner. In the light of these findings L-glutamate or folate cannot be considered as endogenous ligands for hypothetic "kainate receptors".     

Effects of morphine or naloxone on kainic acid neurotoxicity.

Intraperitoneal or subcutaneous administration of kainic acid (KA) (5–15 mg/kg) to adult rats included a syndrome of wef wet dog shakes (WDS), convulsions and brain damage. Components of the syndrome were evoked in a dose-related manner with low doses inducing WDS only and progressively higher doses being associated with an increasing incidence of naloxone (4 mg/kg) 5 minutes prior to KA (12 mg/kg) resulted in a moderate reduction in the incidence of WDS, convulsions and brain damage. Administering morphine (5 or 10 mg/kg) 10 minutes prior to KA (7 mg/kg) markedly enhanced the neurotoxicity of KA as was evidenced in an increase in the incidence of convulsions and brain damage from 7% (KA alone) to 100% (morphine + KA). KA, a structural analog of the putative excitatory transmitter glutamate (Glu), is thought to exert its excitotoxic activity through Glu excitatory receptors. Additional studies are needed to elucidate the mechanism by which morphine and naloxone respectively enhance and suppress KA neurotoxicity and to clarify whether interaction of these agents at either opioid or Glu receptors plays a role in such phenomena.     

Naloxone blocks morphine enhancement of kainic acid neurotoxicity

Kainic acid (KA) is a potent convulsant which, when administered subcutaneously, induces sustained limbic seizures and a pattern of limbic brain damage that is thought to be seizure-mediated. Diazepam suppresses and morphine enhances both the seizures and brain damage induced by KA. Here we show that morphine enhancement of KA neurotoxicity is blocked in a dose-dependent manner by subcutaneous pretreatment with naloxone. Theses and related findings support the hypothesis that morphine enhances the seizure-linked neurotoxicity of KA by an opiate specific action at certain limbic receptor sites where opiates suppress GABAergic activity, thereby lowering the threshold for propagation of seizure activity in limbic circuits.     

Some factors influencing the neurotoxicity of intrastriatal injections of kainic acid

Intrastriatal injections of kainic acid are known to destroy striatal neurons including many containing choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD). Using these enzymes as indices of neuronal loss, the neurotoxicity of small doses of kainic acid was found to be influenced by injection time and volume. It was partly blocked by coninjection of some but not all glutamate antagonists or by prior lesioning of the corticostriatal tract. Other adjuvants, drugs, or lesions tested had little modifying effect, except that changes in the dopaminergic system seemed to increase the toxicity towards cholinergic but not GABAnergic systems. High-affinity glutamate accumulation by neostriatal synaptosomes was significantly increased 1–7 days following kainic acid injections. MAO and acetylcholinesterase activities were depressed in kainic acid-lesioned striata but not nearly as much as were CAT and GAD. An indirect mechanism involving glutamate release and inhibition of reuptake is suggested for kainic acid neurotoxicity.     

3H]kainic acid binding sites in chick cerebellar membranes

1. A total particulate fraction of chick cerebellar membranes, obtained by a simple method, has been found to specifically bind [3H]kainic acid. Non-neuronal tissue, like chick liver, does not show any appreciable specific binding under the same experimental conditions. 2. Specific [3H]kainic acid binding to chick cerebellar membranes increases linearly with tissue concentration, reaches the binding equilibrium almost instantaneously and is pH and temperature dependent. 3. Specifically bound [3H]kainic acid is displaced by suitable concentrations of unlabelled kainic acid, L-glutamic acid and other excitatory amino acid analogues, both agonist and antagonist. This pharmacological pattern agrees with the general pharmacological properties of kainic acid receptors. 4. Saturation kinetic studies of kainic acid binding sites show one single binding mode with an apparent dissociation constant KD = 278 nM and a maximum number of binding sites of 187 pmoles/mg of protein. 5. In view of the mentioned data and the high amount of receptor sites found in chick cerebellar membranes, as compared with related values in rat cerebellum, we suggest that these receptors play a different physiological role or that they have a different cellular localization in chick and rat cerebellum.     

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.     

Huntington's disease and its animal model: alterations in kainic acid binding.

The density of ³H-kainic acid (KA) binding was determined in several regions of Huntington's Diseased (HD) and control human brains. ³H-Kainic acid binding was significantly reduced by 55% in the caudate nucleus and by 53% in the putamen of HD brains. In addition, ³H-KA binding was determined in rat striatum at various intervals following lesion with KA, a procedure which produces an animal model of HD. After KA lesion, ³H-KA binding in the rat striatum underwent a slow reduction, reaching 25% of control after 6 weeks. Several properties of ³H-KA binding to rat brain membranes were also investigated, including inhibition by ions, regional distribution and displacement by various compounds. The findings confirm the validity of the KA-lesioned model for HD and suggest a post-synaptic location for kainic acid receptors in the striatum.     

Prolonged exposure to cigarette smoke blocks the neurotoxicity induced by kainic acid in rats

We examined the effects of cigarette smoke (CS) on three parameters associated with kainic acid (KA)-induced neurotoxicity: seizure activity, cell loss in the hippocampus, and increased Fos-related antigen (FRA) expression. Animals were exposed to the main stream of CS from 15 Kentucky 2R1F research cigarettes containing 28.6 mg tar and 1.74 mg nicotine per cigarette, for 10 min a day, 6 days per week, for 4 weeks, using an automatic smoking machine. KA administration (10 mg/kg, i.p.) produced robust behavioral convulsions lasting 4-5 h. Pre-exposure to CS significantly reduced the seizures, mortality, and severe loss of cells in regions CA1 and CA3 of the hippocampus after KA administration. Consistently, pre-exposure to CS significantly attenuated the KA-induced increased FRA immunoreactivity in the hippocampus. In contrast, pretreatment with central nicotinic antagonist, mecamylamine (2 or 10 mg/kg, i.p.) blocked the neuroprotective effects mediated by CS in a dose-dependent manner. These results indicate that CS exposure provides neuroprotection against the KA insult via nicotinic receptor activation.     

Comparison of the in vitro and in vivo neurotoxicity of three new sources of kainic acid

Summary.  Historically, all commercially available kainic acid has been derived from a single biological source using a consistent method of extraction and purification. That source became unavailable in 1995. Recently, three new commercial suppliers of kainic acid have made the product available, but the source of the material and the purification processes used differ. Our objective was to systematically compare the response produced by each of these new sources of kainic acid using three established neurobiological techniques: neuronal cell culture, hippocampal slice electrophysiology, and whole animal behavioural toxicity. Results in all three systems indicated no overall differences between the three formulations, although studies in both cerebellar neuron cultures and whole animal toxicity testing in mice, revealed some significant differences that may imply subtle differences in receptor selectivity and/or potency. We conclude that all three sources of kainic acid are viable alternatives to traditional kainate but they may not be identical. Until further information becomes available researchers may want to avoid using the three formulations interchangeably, and take note of the source of kainic acid when evaluating literature describing results from other laboratories. Received June 29, 2001 Accepted August 6, 2001 Published online June 26, 2002     

Temporal expression of hippocampal lysophosphatidic acid receptors and their roles in kainic acid-induced neurotoxicity

In this investigation, the role of hippocampal lysophosphatidic acid (LPA) receptors in the regulation of kainic acid (KA)-induced neurotoxicity was investigated. KA (0.07 μg) intracerebroventricular (i.c.v.) administration increased hippocampal Lpar1, 2, 3, and 5 mRNA levels. In the immunohistochemical study, alteration of LPA₁ or LPA₃ immunoreactivity was different depending on the hippocampal regions, such as CA1, CA2, CA3, and dentate gyrus. In addition, the i.c.v. pretreatment with LPA₁ and LPA₃ antagonists, such as VPC12249 (0.05 μg) and VPC32183 (0.05 μg) attenuated KA-induced neuronal cell death in the hippocampal CA3 region. However, the i.c.v. 18:1 LPA (0.05 μg) pretreatment aggravated KA-induced neuronal cell death in the hippocampal CA3 region. Our results suggest that LPA receptors, such as LPA₁ and LPA₃ activation might play an important role in the regulation of KA-induced neuronal cell death in the hippocampal CA3 region.     

Ontogeny of binding sites for [3H]kainic acid in chick and rat cerebellar membranes: A comparative study

We have analyzed the developmental properties of kainate receptors in cerebellar membranes prepared from chick and rat, two vertebrate species with contrasting patterns of functional maturation. Single populations of binding sites have been characterized in the avian and rodent membranes with apparent dissociation constants (K_d) in the 210–280 nM and 40–55 nM ranges, respectively; the number of binding sites (B_max) increases with age in both species, reaching a maximum of 187 pmol/mg in the case of 10-day chicks vs. 1.28 pmol/mg in 75-day rats. The ontogenetic profiles of kainate receptors in chick and rat cerebella are in consonance with the patent differences in motor development at birth.     

Treatment with 7-nitroindazole enhances kainic acid induced cholinergic neurotoxicity in the rat striatum: a neuroprotective role for neuronal nitric oxide

1. In this study we investigated the effect of 7-nitroindazole (7-NI), a preferential inhibitor of neuronal nitric oxide synthase (nNOS), on kainic acid (KA) induced neurotoxicity in rats. Choline acetyltransferase activity (CAT), a cholinergic marker, and histological changes were employed to assess neurotoxicity.2. In control rats, the local intrastriatal injection of 0.5 g of KA reduced CAT from 22.9 ± 2.2 to 14.7 ± 2.0 nmol/h/mg tissue ((38 ± 6)% reduction) (P < 0.001). Greater reductions in CAT were observed with 1 and 2 g of KA ((70 ± 6)% and (80 ± 3)%, respectively). 7-NI aggravated KA-induced cholinergic and histological damage. KA reduced CAT by (68.2 ± 4)% in 7-NI-treated rats, by (38 ± 6)% in saline-treated controls, and by (41 ± 4)% in peanut-oil- (7-NI-vehicle-) treated rats (P = 0.0047).3. After KA, CAT activity averaged 14.3 ± 2.0 in peanut-oil-treated rats and 7.9 ± 1.0 nmol/h/mg tissue in 7-NI- (peanut-oil-) treated rats (P = 0.015). Similarly to changes in CAT, 7-NI treatment aggravated KA-induced histological changes indicative of neuronal damage (acute ischemic neuronal changes, disorganization of myelinated fibers bundle, and vacuolation changes of the neuropil). Treatment with 7-NI was not associated with increased mortality.4. Our findings suggest that neuronal NO plays a neuroprotective action on excitotoxicity.     

Reversible decrease in dopaminergic 3H-agonist binding after 6-hydroxydopamine and irreversible decrease after kainic acid

Six days after the unilateral intrastriatal injection of 30 ug 6-hydroxydopamine (6-OHDA) the number of stereospecific 3H-dopamine and 3H-apomorphine binding sites (Bmax) was reduced by 50-60% in the caudate nucleus ipsilateral to the lesion. The dopamine content of the lesioned caudate nucleus was also reduced to 2% of the contralateral side or of sham-operated controls. The preincubation of depleted homogenates with added dopamine reversed the effects of 6-OHDA on the Bmax of 3H-agonists. A similar pattern of depletion, decrease in binding and in vitro reversal by dopamine was observed after a single injection of reserpine (4.0 mg/kg, im.). The intrastriatal injection of kainic acid also lowered the Bmax of 3H-agonists by 65% without altering dopamine content. Preincubation of homogenates of kainic acid-lesioned caudate nuclei with 355 nM (endogenous) dopamine did not reverse the decrease in binding. We conclude that treatments which deplete endogenous dopamine, including the lesion of nigrostriatal terminals, induce a reversible change in the parameters of 3H-agonist binding whereas the destruction of intrinsic caudate neurons with kainic acid results in an irreversible loss of receptors.     

Quinolinic acid: neurotoxicity

This minireview series reviews some of the most recent findings about quinolinic acid's cellular toxicity and its implications in diseases such as HIV associated neurocognitive disorders, depressive disorders and schizophrenia, and finally therapeutic strategies with drugs able to interfere with quinolinic acid production and/or effects.     

Specific binding of kainic acid to purified subcellular fractions from rat brain

The subcellular distribution of kainic acid (KA) binding sites in rat brain has been studied using a microcentrifugation assay. KA did not bind to myelin or brain cytosol and had few or no binding sites in the nuclear fraction. However, it bound to microsomal components (K _d =128–136 nM; 2.5–4.8 pmol/mg protein), purified synaptic plasma membranes (SPM) (K _d =45–71 nM; 5.8–6.5 pmol/mg), and purified cell-body and intraterminal mitochondria (K _d =11–31 nM; 0.4–1.1 pmol/mg). Bound KA could be totally displaced byl-glutamate orl-aspartate, but several putative antagonists of these amino acids (nuciferin, compound HA-966, 2-amino-4-phosphonobutyrate, and 2-amino-3-phosphonoproprionate) failed to displace KA or did so at very high concentrations (4 mM). Glutamic acid diethyl ester (GDEE) andd,l--aminoadipate (-AA) were more effective (IC₅₀, 0.2–0.8 mM) and showed differential effects in their capacity to displace KA bound to the various subcellular fractions. Thus, GDEE only displaced 40–60% of the KA bound by SPM or mitochondria and did not prevent the binding of KA to microsomes. -AA, on the other hand, was more effective in preventing the binding of KA at high concentrations and displaced between 80 and 100% of the drug. Both compounds showed biphasic curves of KA displacement from synaptic plasma membranes and mitochondria. The overall results indicate the presence of multiple binding sites for KA in brain cells and suggest that KA does not act exclusively at synaptic glutamate receptors. The mechanism of KA action is most likely quite complex, and the drug probably acts at multiple binding sites affecting a number of processes.     

Synthesis of the ω-phosphonic acid analogue of kainic acid

The ω-phosphonic acid analogue of kainic acid was synthesised from the naturally occurring carboxylic acid in nine steps and 6% overall yield.     

Thalamic injections of kainic acid produce myocardial necrosis.

Bilateral injections of 2–5 nmol of kainic acid into the thalamus produce periarteriorlar myocardial necrosis. In addition, gross hematuria is usually observed. Electrolytic lesions of the same area of brain and intracerebral injections of kainic acid at several other locations fail to produce these peripheral changes. Kainic acid at much higher doses subcutaneously or intraperitoneally is also inactive. Urinary noradrenaline levels are increased up to 10-fold during the post-injection period. Some protection against myocardial damage may be produced by reserpine or 6-hydroxydopamine, but atropine seems to confer no protection. The fact that myocardial damage may result from intracerebral lesions and/or pathological stimulation by kainic acid may have clinical implications. Cardiac damage occasionally results in humans from strokes and intracerebral hemorrhage and no satisfactory explanation has ever been offered for the phenomenon of interstitial myocardial fibrosis. The kainic acid model may be one means of studying this phenomenon.     

Distribution and persistence of kainic acid in brain.

The distribution of ³H-kainic acid in rat brain was studied as a function of time after injections of 5 nmoles into the neostriatum, substantia nigra or cerebellum. More than half of the injected material had disappeared from the injection site and the brain by 1/2 hour post injection. Under the conditions used very small amounts of radioactivity (corresponding to less than 7 pmol/ mg of tissue) were found in areas other than the injection site, suggesting that the histological damage reported in the hippocampus and pyriform cortex after striatal injections may be due to a secondary process not dependent on the presence of toxic concentrations of kainic acid in those areas. No radioactivity was found in the TCA-insoluble material nor did it appear that there was rapid metabolism of the bulk of the kainic acid.