Kainic Acid Neurotoxicity Research Articles

In vivo test of two new non-NMDA-receptor antagonist against kainic acid neurotoxicity : M. Berg, T. Bruhn, F.F. Johansen, P. Krogsgaard-Larsen & N.H. Diemer. PharmaBiotec Research Center and Cerebral Ischemia Group, Institute of Neuropathology, University of Copenhagen, Denmark

In vivo test of two new non-NMDA-receptor antagonist against kainic acid neurotoxicity : M. Berg, T. Bruhn, F.F. Johansen, P. Krogsgaard-Larsen & N.H. Diemer. PharmaBiotec Research Center and Cerebral Ischemia Group, Institute of Neuropathology, University of Copenhagen, Denmark

The response of striatal neuropeptide Y and cholinergic neurons to excitatory amino acid agonists

The effect of excitatory amino acid agonists and an antagonist on neuropeptide Y (NPY) and cholinergic neurons in the striatum of the rat was studied by means of NPY immunocytochemistry, DFP histochemistry for acetylcholinesterase (AChE), and biochemical determinations of choline acetyltransferase (ChAT). Intrastriatal infusion of drugs revealed that striatal neurons containing NPY are more sensitive than cholinergic neurons to the neurotoxic actions of kainic acid (KA), quinolinic acid (QA) and l-glutamic acid (GA); all 3 compounds produced a marked loss of NPY neurons, but only a moderate decrease in the number of AChE neurons or ChAT activity. Co-injection experiments showed that the neurotoxicity of QA and GA, but not that of KA, can be antagonized by the specific N-methyl- d-aspartate (NMDA) receptor antagonist 3-((±)-2-(car☐ypiperazine-4-yl))-propyl-1-phosphonic acid (CPP). Destruction of the glutamatergic corticostriatal projection by cerebral decortication protected striatal NPY and cholinergic neurons against KA neurotoxicity. These results indicate that striatal NPY and cholinergic neurons receive prominent cortical amino acid afferents, and that the neurotoxic effect of QA and GA on these neurons is mediated through NMDA receptors.

L-Homocysteic acid: An endogenous excitotoxic ligand of the NMDA receptor

L-Homocysteic acid (L-HCA) has been proposed as a natural transmitter at the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptor based on recent evidence that L-HCA occurs naturally in the mammalian CNS, is released from K + stimulated brain slices in a calcium-dependent manner and may be contained in nerve terminals located in certain brain regions that have a high density of NMDA receptors. Here we report that L-HCA potently induces a pattern of cytopathology in the ex vivo chick retina which mimics the pattern of NMDA but not kainic acid (KA) neurotoxicity. We also show that known NMDA antagonists, including Mg ++, D-aminophosphonopentanoate and certain anesthetics, analgesics, and sedative hypnotics block the neurotoxic actions of L-HCA in direct proportion to their efficacy in blocking NMDA neurotoxicity. While there is a perfect correspondence between agents that block NMDA and L-HCA neurotoxicity, only a few such agents are active against KA neurotoxicity. We find that 3H-Glu binding is inhibited more potently by L-HCA ( Ki =9.4 μM) than NMDA ( Ki=67 μM). Moreover the patterns with which L-HCA and NMDA displace 3H-Glu binding in autoradiograms appear essentially identical. These findings are consistent with the proposal that L-HCA is an endogenous ligand at NMDA receptors.

Neuropathological changes in the rat brain cortex in vitro: effects of kainic acid and of ion substitutions

The ionic mechanisms that may contribute to the neurotoxicity of kainic acid, were studied in a system of rat thin neocortical slices superfused in vitro. Slices superfused for 3 h under control conditions showed an essentially normal aspect when studied by light microscopy. Presence of 30 μM kainate in the superfusion fluid induced neuronal swelling, nuclear condensation and signs of necrosis in some cells, while other neurons, especially in deeper layers, appeared dark and condensed, with microvacuolation. The neuropil presented numerous profiles of swollen dendrites. When the slices were superfused with chloride-free medium, a large number of pyknotic neurons was seen. This was further enhanced by 30 μM kainate, which produced no swelling in this medium. These effects of Cl-free medium were almost entirely prevented in Cl-free medium without calcium and with 0.1 mM of EGTA. Sodium-free medium induced a marked neuronal swelling that was not much changed by kainate. When calcium in an otherwise normal superfusion fluid was reduced to 0.1 mM, a large number of pyknotic neurons, some with incrustations, were seen. Kainate (30 μM) in this low calcium medium led to a very large swelling and destruction of neurons, and to a spongy neuropil. These effects of kainate were greatly intensified in calcium-free-EGTA (0.1 mM) medium. Ca-free-EGTA medium by itself induced considerable neuronal and neuropil swelling. It is concluded that kainate induces neuronal swelling by a sodium- and chloride-dependent mechanism, and the enhancement of swelling in low calcium is due to an increased sodium uptake. Neuronal pyknosis, on the other hand, is induced by seizure-inducing conditions such as Cl-free medium, kainate, 0.1 mM calcium, or 1 mM pentylenetetrazol, apparently in a calcium-dependent manner. It is postulated that neuronal condensation and pyknosis are due to a calcium-induced cytoskeletal collapse.

Inhibition of [3H]kainic acid receptor binding by divalent cations correlates with ion affinity for the calcium channel

Since the neurotoxicity of kainic acid may be due to the opening of membrane channels for calcium ions for (Ca2+), the effects of Ca2+ and other cations were examined on the specific binding of [3H]kainic acid to membranes from the forebrain of the rat. [3H]Kainic acid bound to a high affinity site (KD = 5.6 nM) that was inhibited in a concentration-dependent manner by Ca2+ ions with an IC50 of 3.2 mM. In the presence of 1 mM Ca2+, the KD of the binding of [3K]kainic acid increased to 11.1 nM without any change in the Bmax. The divalent cations, manganese and cobalt, also were potent inhibitors of the binding of [3H]kainic acid, while barium and strontium were much weaker. The inhibitory effects of Ca2+ on the binding of [3H]kainic acid were blocked by the inorganic Ca2+ channel blockers, cadmium and lanthanum. These data suggest that Ca2+ modulates the binding affinity [3H]kainic acid through an allosteric interaction between the binding site on the Ca2+ channel and the kainic acid receptor.

Intrahippocampal injection of kainic acid produces significant pyramidal cell loss in neonatal rats

Previous reports 655 have indicated that pyramidal cells in the developing rat hippocampal formation are not destroyed by intraventricular or inlraperitoneal administration of kainic acid. We examined the neurotoxic properties of kainic acid and ibotenic aeid following intrahippocampal injection in neonatal rats and found significant pyramidal cell death following injection of 1.0 μg kainic acid in 6, 7 and 9-day-old pups. At doses 2.5 or live times this amount, significant pyramidal cell loss was obtained in 5-day-old rats as well. The susceptibility of pyramidal neurons to kainic acid increased as a function of age. The developing hippocampus was considerably more vulnerable to ibotenic acid compared with kainic acid, in contrast to the order of potency reported in adult rats. The increased sensitivity of CA3 pyramidal cells parallels the development of the mossy fiber innervation to the dendrites of these cells supporting the twofold mechanism suggested by Coyle for kainic acid neurotoxicity; that is, a direct cytotoxic action via postsynaptic receptors as well as increased sensitivity due to the presence of excitatory inputs.

Kainic acid neurotoxicity: Characterization of blood-brain barrier damage

The alterations in the water, sodium (Na) and potassium (K) contents of the frontoparietal cortex, hippocampus and thalamus as well as the protein permeability of the blood-brain barrier were investigated in rats 4 h after systemic kainic acid administration. Increases in the water and Na contents and a decrease in the K content were observed together with Evans blue extravasation in the thalamus area indicating the development of vasogenic brain edema. Changes observed in the ion contents of the frontoparietal cortex and hippocampus may be due to a general cell membrane permeability damage but are not caused by a primary disturbance of the blood-brain barrier.

The mechanism of kainic acid neurotoxicity (reply)

The mechanism of kainic acid neurotoxicity (reply)

Neurochemical, pharmacological, and developmental studies on cerebellar receptors for dicarboxylic amino acids.

Specific binding of L-[3H]glutamate ([3H]Glu) and L[3H]Asp) to cerebellar membranes represented a time-, temperature-, pH- and protein-dependent interaction which was both saturable and reversible. Binding sites for both radioligands appeared maximally enriched in synaptosomal fractions isolated by gradient centrifugation. Kinetically derived dissociation constant (K off/K on = Kd) for [3H]Glu binding to this fraction indicated high-affinity (433 nM). Competition experiments employing analogs of excitatory amino acids, including new antagonists, helped identify binding sites for [3H]Glu and [3H]Asp as receptors with differential pharmacological specificities. Membrane freezing reduced numbers of both receptor types, but binding activity could be recovered partially by incubation at 37 degrees C. Glu receptors exhibited a pronounced deleterious sensitivity to thiol modifying reagents and L-Glu (50-1000 microM) provided protection against these compounds during co-incubation with cerebellar membranes. It is suggested that cold storage may induce partially reversible receptor inactivation by promoting sulfhydryl group/bond modification. Rat cerebellar glutamatergic function (endogenous Glu content, Glu uptake and receptor sites) exhibited an apparent ontogenetic peak between days 8-12 postpartum with a plateauing profile from day 30 to adulthood. The accelerated development (days 8-12) coincides with the first demonstrable Glu release and kainic acid neurotoxicity, as described previously.

The mechanism of kainic acid neurotoxicity.

The putative excitatory transmitters glutamate and aspartate, as well as their excitatory analogues, can kill neurones in the central nervous system and may thus be involved in the pathogenesis of various neurodegenerative disorders. Several studies have suggested that postsynaptic receptors are important in the mechanism of toxicity. However, presynaptic factors might also be involved because, in some brain areas, the neurotoxicity of kainate (a potent structural analogue of glutamate) is greatly reduced following elimination of afferent excitatory innervation, even though the postsynaptic excitatory potency of kainate may be unaltered in these conditions. The supply of glutamate from the afferent nerve endings has been suggested to be a necessary factor. Recently, Ferkany, Zaczec and Coyle concluded from studies on slices of mouse cerebellum that kainate activates presynaptic kainate receptors on parallel fibre terminals to release glutamate and that it is the postsynaptic interaction between kainate and the released amino acid that is instrumental in causing neuronal necrosis. The more direct evidence we report here does not support these conclusions.

Pharmacological manipulation of GABA system does not protect the goldfish optic tectum from the neuroexcitatory and neurotoxic action of kainic acid

Inhibition of GABA transaminase which led to a several-fold increase of GABA levels in the goldfish optic tectum or diazepam pre-treatment, were unable to protect tectal neurons from kainic acid neurotoxicity, as judged by light and electron microscopic observations and by the drop of marker enzymes for neurotransmitters. In an in vitro preparation of tectal slices GABA, added to the incubation medium, had no effect on a metabolic parameter (CO 2 production from exogenous glucose) related to the excitatory action of kainic acid. It is concluded that, in the goldfish optic tectum, pharmacological manipulation cannot enhance the activity of GABAergic circuits to the extent necessary to block the neuroexcitatory and neurotoxic action of kainic acid.

Evidence for interactions between [ 3H]glutamate and [ 3H]kainic acid binding sites in rat striatal membranes. Possible relevance for kainic acid neurotoxicity

In vitro studies have shown that kainic acid (10 −6 M) but not N-methyl- d-aspartate (NMDA) in the same concentration reduces the number of striatal [ 3H]glutamate binding sites and increases their affinity in striatal membranes. In vitro studies also show that l-glutamate (10 −8 M) but not NMDA (10 −6 M) increases the number of [ 3H]kainic acid binding sites and reduces their affinity in striatal membranes. Ibotenic acid (10 −6 M) can also reduce the affinity of [ 3H]kainic acid binding sites in striatal membranes. These results give indications for the existence of bidirectional receptor-receptor interactions between two receptors for excitatory amino acids in local striatal circuits. These interactions could partly explain the involvement of glutamate in kainate neurotoxicity.

α-Amino-ω-phosphono carboxylates block ibotenate but not kainate neurotoxicity in rat hippocampus

(−) 2-Amino-7-phosphonoheptanoate and (−) 2-amino-5-phosphonovalerate were shown to possess selective and powerful antagonistic activity vis-à-vis the neurotoxic effects of ibotenic acid in the rat hippocampal formation. The neurotoxicity of kainic acid was not blocked by either drug.

Neurotoxic effect of kainic acid on ultrastructure and gabaergic parameters in the goldfish cerebellum

Kainic acid administration into the cerebellar dorsal lobe of the goldfish causes selective degeneration of some neuronal types. Stellate and Golgi neurons are very sensitive to the neurotoxin and undergo rapid degeneration. On the basis of their differential response to kainic acid, Purkinje cells can be divided in two distinct sub-populations (i.e. sensitive and insentitive neurons). The degenerative changes of the Purkinje neurons are in addition remarkably slow in comparison with the same cells in mammals or with stellate and Golgi neurons in the goldfish. Granule cells, as well as the cerebellar afferent fiber system, are not significantly affected. Six days after kainic acid administration, the level of glutamate decarboxylase in the cerebellar dorsal lobe drops to about 40% of the control value. This result suggests that the neurons sensitive to kainic acid neurotoxicity are, at least in part, GABAergic. Light- and electron-microscopic autoradiography of cerebellar elements selectively accumulating [ 3H]GABA, supports this idea. Moderate decreases of acetylcholinesterase and protein content were also noticed in the kainic acid-treated cerebellar dorsal lobe.

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. These 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.

Systemic injection of kainic acid: Effect on neurotransmitter markers in piriform cortex, amygdaloid complex and hippocampus and protection by cortical lesioning and anticonvulsants

Systemic injection of kainic acid (12 mg/kg) in rats induces a well established pattern of neuronal lesions in different brain regions. These lesions are accompanied by changes in neurotransmitter markers. In the piriform cortex and amygdaloid complex, the kainic acid lesion was accompanied by a reduction in the high affinity uptake of glutamate and in the activities of glutamate decar☐ylase and choline acetyltransferase, whereas in the hippocampus there was a reduction in the high affinity uptake of glutamate and in glutamate decar☐ylase activity. Hemidecortication, hemitransection, a caudal knife cut in the cortex, or treatment with diazepam, all protected against the effects of kainic acid in the piriform cortex and amygdaloid complex but not in the hippocampus. Diphenylhydantoin had no effect on the neurotoxicity of kainic acid. The results indicate that the neurotoxic effects of kainic acid in the piriform cortex and amygdala are dependent on an intact cortical structure, probably due to a dependence on specific excitatory circuitry. The neurons involved may be glutamergic/aspartergic.

Homotaurine (3 aminopropanesulfonic acid; 3APS) protects from the convulsant and cytotoxic effect of systemically administered kainic acid.

Pretreatment of rats with homotaurine (3 aminopropanesulfonic acid; 3APS), a synthetic gamma-aminobutyric acid (GABA) analog, protected from the convulsant and cytotoxic action of systemically injected kainic acid (KA). Wet dog shaking (WDS) behavior was significantly reduced. Taurine, an inhibitory non-GABA-mimetic amino acid, and muscimol (another direct GABA-agonist) reduced the number of seizures and lesions in the brain but were less effective than homotaurine. Progabide (a GABA-agonist) did not modify kainic acid effects. The neurotoxicity of kainic acid could have been due to repetitive convulsive activity. Activation of GABA-mediated inhibition is an effective, but not the determinant means of preventing KA-induced abnormalities.

Kainic acid neurotoxicity; effect of systemic injection on neurotransmitter markers in different brain regions

Systemic injection of kainic acid (12 mg/kg) induces necrosis and neuronal degeneration in several brain regions. The most pronounced effects were observed in the piriform cortex, amygdaloid complex, hippocampus and septum. A good correlation between morphological changes and changes in some neurotransmitter markers was observed in these 4 areas. High affinity uptake of L-glutamate, as well as glutamate decarboxylase and choline acetyltransferase activities were reduced in the piriform cortex and amygdaloid complex whereas in the hippocampus and septum only the first two markers were reduced. No morphological changes or decrease in any of these neurotransmitter markers were observed in striatum or globus pallidus. A pronounced neuronal degeneration could be demonstrated in lateral thalamus and geniculate body, but this degeneration was not accompanied by any decrease in the transmitter markers tested.

Kainic acid neurotoxicity does not depend on intact retinal input in the goldfish optic tectum

Kainic acid neurotoxicity has been studied in the optic tectum of the goldfish 4 eeks after eye enucleation. The effect of drug treatment has been tested with respect to both neurochemical and morphological parameters. The neurotransmitter-related enzymes, choline acetyltransferase, acetylcholinesterase and glutamate decar☐ylase, show about 50% decrease in the deafferentated tectum 6 days after kainic acid administration. Relevant morphological alterations of the tectal structure can also be noticed at the same stage. The neurotoxic effects of kainic acid in the deafferentated optic tectum are therefore quite similar to the effects previously noticed for the intact optic tectum of normal fish. Control experiments on the effect of optic nerve degeneration by itself on the levels of the neurotransmitter-related enzymes in the optic tectum, have shown no significant decrease in glutamate decar☐ylase, a slight decrease in acetylcholinesterase and a more marked drop in choline acetyltransferase. The findings are discussed with reference to some of the hypotheses advanced in order to explain kainic acid neurotoxicity. It is proposed that the neurotoxic effect of kainic acid after removal of specific excitatory afferents, may vary in differrent nervous centers depending on differences of the remaining extrinsic connections and of the intrinsic neural circuits.

Dopamine receptor stimulation and striatal kainic acid neurotoxicity.

Dopamine receptor stimulation and striatal kainic acid neurotoxicity.