Robert Zaczek

Excitatory amino acid analogues: neurotoxicity and seizures.

The neurotoxic and convulsant properties of conformationally restricted and synthetic analogues of excitatory acidic amino acids were examined after stereotaxic injection into the striatum and the dentate gyrus of the hippocampal formation. In the striatum, neurotoxicity was quantified by the reduction in the activity of choline acetyltransferase and glutamate decarboxylase, markers for striatal intrinsic neurons. The following sequence of neurotoxic potencies was defined; kainic acid approximately equal to domoic acid much greater than alpha-keto kainic acid approximately equal to alpha-allo kainic acid greater than ibotenic acid approximately equal to cis-cyclopentyl glutamic acid greater than quisqualic acid approximately equal to N-methyl-D-aspartic acid. When normalized for neurotoxic potencies, a wide variation in the convulsant effects of the agents was observed after hippocampal injection. N-Methyl-D-aspartate produced nearly continuous electroencephalographic seizures for 2 hr after injection, where alpha-keto-kainate and kainate and quisqualate caused seizure activity for 64 and 45% respectively of this period; kainate, alpha-allo kainate and domoate caused intermittent seizure activity during approximately 30% of the recording period; ibotenate and cyclopentylglutamate had minimal convulsant effects. Seizures were associated with a significant reduction in the levels of norepinephrine and with increases in the levels of 5-hydroxyindoleacetic acid in the cortex and hippocampal formation and increases in the levels of gamma-aminobutyric acid in the hippocampal formation. Kainate, domoate, keto-kainate and alpha-allo-kainate caused extensive lesions of the hippocampal formation that also involved the pyriform cortex; ibotenate and cyclopentylglutamate caused uniform but substantial lesions limited to the dentate gyrus, whereas quisqualate and N-methyl-D-aspartate produced small and restricted lesions. The results demonstrate a poor correlation between the neurotoxic and convulsant potencies of these excitatory amino acid analogues and suggest that receptor-specific interactions may account for these disparities.

Microinjection of kainic acid into the rat hippocampus

The effects of unilateral injection of kainic acid into the rate hippocampus have been examined in terms of morphologic, neurochemical and behavioral sequelae. Infusion of 10 nmoles if kainate causes a rapid and complete degeneration of neuronal perikarya in the entire hippocampal formation followed by gliosis and atrophy of the region. This unilateral neuronal loss is accompanied by a 50% decrease in the specific activity of the biochemical markers for GABAergic neurons including glutamic acid decarboxylase, endogenous GABA and synaptosomal uptake of [3H]GABA. The extrinsic hippocampal cholinergic and noradrenergic afferents also exhibit significant alteration. Although the specific activity of choline acetyltransferase is unaffected and the specific activity of tyrosine hydroxylase is significantly increased in the injected hippocampus, the synaptosomal high affinity uptake process for [3H]choline and [3H]norepinephrine are significantly reduced at 10 days after injection. Whereas the level of endogenous acetylcholine is elevated in the lesioned hippocampus at 2 days after injection, the level of endogenous norepinephrine is reduced. For several hours after intrahippocampal injections of 5 nmoles or more of kainate, rats exhibit epileptiform behavior. Intrahippocampal injection of kainate may be a useful rodent model for temporal lobe seizure disorders.

Local and Distant Neuronal Degeneration Following Intrastriatal Injection of Kainic Acid

After intrastriatal injection, the neurotoxin, kainic acid, was cleared from the rat forebrain in a biphasic manner with 70% eliminated within 2 hours; by 24 hours after infusion, less than 1% of the kainic acid remained in the forebrain. The kainic acid diffused into adjacent brain structures, achieving mu molar concentrations in several regions ipsilateral to the injected striatum. At various times after intrastriatal injection of 9.3 nmoles of kainic acid, the brain was serially sectioned; the sections were stained for Nissl substance with cresyl violet or for degenerating neurons with the ammoniacal silver method. Neuronal degeneration spread unevenly into contiguous structures from the central sphere in the injected striatum and affected the ipsilateral pyriform cortex and amygdala, the deep layers of the overlying cerebral cortex, and the medial aspects of the bed nucleus of the stria terminalis and of the nucleus accumbens. In half of the rats, the pyriform cortex contralateral to the side of injection also underwent degeneration. A subpopulation of pyramidal cells in layer IV of the lateral neocortex and the CA3-CA4 pyramidal cells in the ipsilateral hippocampus were selectively affected, whereas adjacent neurons remained intact. The distribution of agyrophilic fibers and terminals in subcortical structures was consistent with the degeneration of neurons of origin in the affected striatal and extrastriatal regions. Brain sections stained by the gold sublimate technique from rats perfused 20 days after injection revealed an intense astrocytic response in all areas affected by acute neuronal degeneration. Extrastriatal damage could be markedly reduced by injection of lower doses of kainic acid (2.3 nmoles) with brief anesthesia; under these conditions, however, the subpopulation of large striatal neurons were relatively resistant, as compared to the Golgi II neurons. These studies demonstrate significant and variable neuronal degeneration beyond the primary site of the lesion after intracerebral injection of kainic acid; several factors affect the pattern of degeneration, including the amount of kainic acid injected, its biological activity, its diffusion, duration of anesthesia, and variable sensitivity of neurons. Consequently, care must be exercised in the use of this neurotoxin to determine the extent and selectivity of neuronal damage, particularly with reference to neuronal vulnerability beyond the central sphere of intrinsic neuronal degeneration.

Effects of anaesthetics and anticonvulsants on the action of kainic acid in the rat hippocampus

The effects of anaesthetics and anticonvulsants on the acute behavioral response and neurochemical alterations following intrahippocampal injection of kainic acid were examined. When compared to the effects in animals anaesthetized with a combination of chloral hydrate and pentobarbital (Equithesin), brief anaesthesia with ether or with hexobarbital potentiated whereas prolonged anaesthetia attenuated the action of 0.5 microgram of kainic acid. Of the constituents of Equithesin, chloral hydrate offered less protection than pentobarbital. The anticonvulsants, phenobarbital, diazepam, diphenylhydantoin, and carbamazepine offered partial protection when administered in conjunction with ether anaesthetia.

Long-term sequelae of striatal kainate lesion

Injection of the conformationally restricted analogue of glutamate, kainic acid, into the striatum causes a profound degeneration of neurons intrinsic to the region while sparing several identified types of axons of passage or of termination arising from outside the striatum 6,v,x2,14. The striatal kainate lesion is accompanied by a marked reduction in the presynaptic markers for cholinergic and GABAergic neurons. In contrast, the presynaptic markers for the dopaminergic and serotonergic terminals innervating the region are not reduced 14,~5. Whereas the lesion causes a 40~, reduction in the density of muscarinic cholinergic receptors, GABA receptor binding exhibits a 3-fold increase, reflecting an enhanced affinity of the receptor for its ligand 3,14. Although the degeneration of the intrinsic neurons takes place within 48-72 h 6, secondary changes, including axonal atrophy due to loss of postsynaptic sites, axonal sprouting and alterations of the neurochemical characteristics of the remaining neuronal processes, would presumably occur much more gradually. Accordingly, we have examined the neurochemical and morphologic sequelae of the kainate lesion to the rat striatum 9 months after injection of the neurotoxin. Sprague Dawley rats (160 g) received a stereotaxic injection of 10 nmole of kainic acid (Sigma Chemicals, St. Louis, Mo., Lot 105C-0064) in the right striatum as previously described 14. After the acute period of aphagia and adipsia, the rats were maintained under standard feeding and lighting conditions in the animal quarters for nine months. For assays of neurotransmitter-synthesizing enzymes, the animals were decapitated and the injected and contralateral striata were removed, homogenized in 50 mM Tris.HCl buffer, pH 7.4, containing 0.2 ~ Triton X-100. The activities of tyrosine hydroxylase, choline acetyltransferase and glutamic acid decarboxylase were assayed 2,4, 16. For measurement of the synaptosomal high affinity uptake processes, the animals were decapitated and the injected and contralateral striata were immediately dissected at 5 °C, homogenized in 20 vols of 0.3 M sucrose buffered with 50 mM of Tris.HCl, pH 7.4; the P2 fraction isolated by differential centrifugation was washed and resuspended in 20 vols of the homogenizing buffer. Aliquots of the resuspended P2 fraction were incubated for 4 min at 25 °C with 1 /zM [aH]GABA and at 37 °C with 0.05/~M

Characteristics of chloride-dependent incorporation of glutamate into brain membranes argue against a receptor binding site

Although membrane sites from brain, labelled with [3H]glutamate (Glu) under sodium-free conditions, are thought to represent excitatory receptors, certain anomalous characteristics of the kinetics of apparent binding raised the question of whether transport might contribute to this process, prompting a closer examination of it. Hyperosmolar media and low incubation temperatures (4 degrees C) both led to decreases in the apparent specific binding of [3H]glutamate to membranes from the brain of the rat in the presence of chloride. Furthermore, only 15% of the [3H]glutamate, bound at 37 degrees C, was dissociable when the membranes were then cooled to 4 degrees C. The binding of [3H]glutamate was increased in the presence of certain dipeptides such as L-phenylalanyl-L-glutamate (Phe-Glu); and the binding augmented by the presence of Phe-Glu, was also sensitive to temperature and osmolarity of the incubation buffer. Sonication of membranes in 5 mM glutamate increased the apparent binding of [3H]glutamate and abolished the stimulatory effect of Phe-Glu. These findings are consistent with the hypothesis that chloride-dependent association of [3H]glutamate with membranes from brain reflects, in part, a sequestration process, which may be driven by glutamate exchange.

Kainic acid neurotoxicity and seizures

Abstract The effects of anesthetics and anticonvulsants on the neurotoxic effects of kainic acid (2.3 nmol) injected into the rat hippocampus and striatum have been examined with regard to neurochemical, histological and electroencephalographic alterations. Brief anesthesia with ether resulted in seizures and severe degeneration of the hippocampal CA1−4 pyramids and granule cells. Prolonged anesthesia (> 300 min) with chloral hydrate-pentobarbital or with γ-butyrolactone protected most hippocampal neurons and blocked seizures. Phenobarbital (100 mg/kg) prevented cortical seizures but provided minimal protection against hippocampal neuronal degeneration. Prolonged anesthesia (> 300 min) with chloral hydrate-pentobarbital also attenuated the neurotoxic effects of kainic acid in the striatum. The anesthetic and not the anticonvulsant action of drugs accounted for the protection against the neurotoxicity of kainic acid at the site of injection.

N-methyl-d-aspartic acid: A convulsant with weak neurotoxic properties

N-Methyl-D-aspartic acid (NMDA) is 100-fold less potent as a neurotoxin than kainic acid when injected into the rat striatum. However, NMDA, when injected into the hippocampus, causes a more severe seizure disorder than kainic acid and doses of NMDA than produce much smaller lesions than those caused by kainate. These results indicate a poor correlation between convulsant and neurotoxic properties of acidic excitatory amino acids.

Glutamate-containing dipeptides enhance specific binding at glutamate receptors and inhibit specific binding at kainate receptors in rat brain

The dipeptide, L-phenylalanyl-L-glutamate (PG), augments the specific binding of the excitatory amino acid receptor antagonist, [3H]2-amino-7-phosphonoheptanoic acid (APH), to rat forebrain membranes by 5-fold at 100 microM with an EC50 of 4.9 microM. The increase in the specific binding of [3H]AHP induced by PG results exclusively from an increase in Bmax. In contrast, PG inhibits the specific binding of [3H]kainic acid to forebrain membranes with a Ki of 6.8 microM. Of several related peptides examined, active ones affected the two receptor sites in a reciprocal fashion. The results suggest an allosteric interaction between [3H]APH and kainate receptors modulated by glutamate-containing peptides.

A simple method for quantitative electroencephalographic assessment of drugs with convulsant and anticonvulsant properties

A simple, inexpensive and reliable microcomputer-assisted method to detect and quantify abnormal EEG spiking is described. High frequency wave forms (20-40 Hz) with high amplitude are discriminated using a beta-2 bandpass filter and a threshold comparater. The spikes are then compiled and reported by an Apple II+ microcomputer. The method was validated by measuring seizures generated by intraperitoneal injection of pentylenetetrazol (PTZ), by microinjection of excitatory amino acids into the dorsal hippocampus, and by the antagonism of these seizures by diazepam and 2-amino-7-phosphono heptanoic acid, respectively.