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.     

Light-Stimulated Release of Taurine from Retinas of Kainic Acid-Treated Chicks

Abstract: The light-stimulated release of [³H]taurine from chick retina was studied in chicks intraocularly injected with kainic acid (60 nmol). This treatment produced a loss of more than 80% of the inner nuclear and the inner synaptic layers, sparing the outer retinal layers. Concomitantly, the treatment produced a marked decrease of endogenous GABA and glycine but not of taurine. The activity of glutamate decarboxylase was also markedly decreased in the kainic acid-treated retinas. The release of [³H]taurine, either spontaneous or stimulated by light, was unaffected by the treatment. These results suggest that the light-stimulated efflux of taurine occurs from the retinal layers which are not affected by the kainic acid treatment.     

Changes in Extracellular Amino Acids During Soman-and Kainic Acid-Induced Seizures

Extracellular amino acid levels in the rat piriform cortex, an area highly susceptible to seizure-induced neuropathology, were determined by means of intracranial microdialysis. Seizures were induced by systemic administration of either soman (O-1,2,2-trimethylpropyl methylphosphonofluoridate), a potent inhibitor of acetylcholinesterase, or the excitotoxin kainic acid. Extracellular glutamate levels increased in animals with seizures shortly after administration of either convulsant, but this change was statistically significant only in the case of soman-treated animals. Extracellular taurine levels increased markedly, reaching two- and fourfold baseline levels during the second hour of soman- and kainic acid-induced seizures, respectively. Taurine levels did not increase in the subpopulation of soman-treated animals without seizures, a finding indicating that elevation of extracellular taurine level is seizure related. Thus, we propose that taurine efflux may be a physiological cellular response to neuronal changes produced by excitotoxic chemicals, either directly or as a consequence of seizures.     

2-Amino-4-Phosphonobutyric Acid Exerts a Light-Dependent Effect on Post-Gabaculine Levels of Retinal γ-Aminobutyric Acid (GABA): Evidence that ON Synaptic Pathways Regulate Retinal GABAergic Transmission

Abstract: The effects of light, 2-amino-4-phosphonobutyric acid (APB), and kainic acid on rat retinal γ-aminobutyric acid (GABA)-ergic transmission were studied by measuring levels of retinal GABA following subcutaneous injection of gabaculine, an irreversible inhibitor of GABA-transaminase. Post-gabaculine levels of retinal GABA in light-exposed rats were significantly greater than those in rats held in darkness. The synaptic mechanism of this effect of light was examined by measuring post-gabaculine levels of retinal GABA in rats placed into either lighted or darkened conditions after receiving unilateral intravitreal injections of APB, a glutamate analogue that selectively decreases the activity of ON synaptic pathways in the retina. APB attenuated the post-gabaculine accumulation of GABA in rats held in the light, but not in those placed into darkness. Furthermore, the light-dependent increment in post-gabacu line accumulation of retinal GABA was entirely APB sensitive, and the effect of APB was entirely light dependent. In contrast to APB, kainic acid stimulated the post-gabaculine accumulation of retinal GABA in vivo. Our findings suggest that APB and kainic acid influence GABAergic transmission at different sites in the retina and that some retinal GABAergic neurons are either ON or ON-OFF amacrine cells.     

Gonadal hormones affect neuronal vulnerability to excitotoxin-induced degeneration

The role of endogenous gonadal secretions in neuroprotection has been assessed in a model of hippocampal degeneration induced by the systemic administration of kainic acid to adult male and female rats. A low dose of kainic acid (7 mg/Kg b.w.) induced a significant loss of hilar dentate neurons in castrated males and did not affect hilar neurons in intact males. The effect of kainic acid on hilar neurons in female rats was different depending on the day of the estrous cycle in which the neurotoxin was administered; while no significant effect of kainic acid was observed when it was injected in the morning of estrus, there was a significant loss of hilar neurons when it was injected in the morning of proestrus as well as when it was injected into ovariectomized rats. Estradiol or estradiol plus progesterone prevented hilar neuronal loss when injected simultaneously with kainic acid in ovariectomized rat. Progesterone by itself did not prevent neuronal loss induced by kainic acid and estogen was only effective when it was injected either 24 h before or simultaneously with kainic acid and not when it was injected 24 h after the administration of the toxin. These findings indicate that endogenous gonadal hormones protect hippocampal hilar neurons from excitotoxic degeneration. In addition, the timing of exposure to ovarian hormones and the natural fluctuation of ovarian hormones during the estrous cycle may influence the vulnerability of hilar neurons to excitotoxicity. These findings are relevant to possible modifications in neurodegenerative risk in humans as endogenous levels of gonadal hormones change during the menstrual cycle and during aging.     

Possible destruction of cholinoceptive neurons by overstimulation of cholinergic tracts

It is well established that intracerebral injections of kainic acid may cause not only neuronal cell destruction at the injection site, but also losses in some distant regions. The mechanisms are different. The distant, but not the local, destruction can be produced by folic as well as by kainic acid and prevented by pretreatment of the animal with diazepam. Overexcitation of excitatory projections is believed responsible for the distant damage and evidence is presented that in some instances the projections involved are cholinergic. Thus, for example, injections of kainic acid or folic acid into the substantia innominata of rats destroy neurons in areas such as the pyriform cortex and amygdala which receive cholinergic projections from the injected area. Some of the destroyed neurons are GABAergic. That the distant toxicity in these areas can be partially blocked by scopolamine and is accompanied by decreases in the number of muscarinic binding sites is consistent with a cholinergic mechanism. Distant damage also occurs in the thalamus but this appears to be mediated by a noncholinergic projection. Similar injections of folic acid or kainic acid into the rostral pontine tegmentum, another area with cholinergic cells, cause destruction of both dopaminergic and GABAergic neurons in the substantia nigra. The effect on the GABAergic but not that on the dopaminergic cells is blocked by scopolamine. The results are discussed in relation to possible mechanisms of epilepsy and of selective neuronal losses in diseases such as Parkinson's disease.     

Ganglion cells of chicken retina possess nicotinic rather than muscarinic acetylcholine receptors

Chicken retinas were exposed to intravitreal kainic acid to destroy amacrine and bipolar cells at low concentrations, and horizontal cells at high concentrations in addition. Ganglion cells were destroyed by intravitreal injections of colchicine. Low doses of kainic acid reduced the number of binding sites for both [³H]quinuclidinyl benzilate (muscarinic acetylcholine receptors) and N-[propionyl ³H]-bungarotoxin (nicotinic acetylcholine receptors), with little additional loss at higher doses. In contrast, colchicine reduced the number of binding sites for N-[propionyl-³H]-bungarotoxin, but had little or no effect on the number of binding sites for [³H]quinuclidinyl benzilate. These results are consistent with the idea that, in chicken retina, cholinergic amacrine cells make contact with ganglion cell dendrites at sites which possess mainly nicotinic acetylcholine receptors, while both types of receptor are involved in interactions between amacrine cells and perhaps bipolar cells.     

ACTION OF THE NEUROTOXIN KAINIC ACID ON HIGH AFFINITY UPTAKE OF l-GLUTAMIC ACID IN RAT BRAIN SLICES

Kainic acid is a linear competitive inhibitor (K_is 250 μm ) of the ‘high affinity’ uptake of l -glutamic acid into rat brain slices. Kainic acid inhibits the ‘high affinity’ uptake of l -glutamic, d -aspartic and l -aspartic acids to a similar extent. Kainic acid is not actively taken up into rat brain slices and is thus not a substrate for the ‘high affinity’ acidic amino acid transport system or any other transport system in rat brain slices. Kainic acid (300 μm ) does not influence the steady-state release or potassium-stimulated release of preloaded d -aspartic acid from rat brain slices. Kainic acid binds to rat brain membranes in the absence of sodium ions in a manner indicating binding to a population of receptor sites for l -glutamic acid. Only quisqualic and l -glutamic acid inhibit kainic acid binding in a potent manner. The affinity of kainic acid for these receptor sites appears to be some 4 orders of magnitude higher than for the ‘high affinity’l -glutamic acid transport carrier. Dihydrokainic acid is approximately twice as potent as kainic acid as an inhibitor of ‘high affinity’l -glutamic acid uptake but is some 500 times less potent as an inhibitor of kainic acid binding and at least 1000 times less potent as a convulsant of immature rats on intraperitoneal injection. Dihydrokainic acid might be useful as a ‘control uptake inhibitor’ for the effects of kainic acid on ‘high affinity’l -glutamic acid uptake since it appears to have little action on excitatory receptors. N-Methyl-d -aspartic acid is a potent convulsant of immature rats, but does not inhibit kainic acid binding or ‘high affinity’l -glutamic acid uptake. N-Methyl-d -aspartic acid might be useful as a ‘control excitant’ that activates different excitatory receptors to kainic acid and does not influence ‘high affinity’l -glutamic acid uptake.     

IV. Neurotoxicity of colchicine and other tubulin-binding agents: A selective vulnerability of certain neurons to the disruption of microtubules

Colchicine and certain other agents which disrupt microtubules and interfere with axonal and dendritic transport are highly toxic to certain CNS neurons. The present chapter summarizes our knowledge about this selective neurotoxicity. Injections of colchicine into several brain regions lead to the death of selected populations of neurons within those regions. Intra-hippocampal injections selectively destroy granule cells of the dentate gyrus; hippocampal pyramidal cells are essentially unaffected. Injections into the cerebellum, olfactory bulb, and caudate nucleus also destroy resident neurons. In these areas several cell types are vulnerable. Neurons of the cerebral cortex appear to be much less affected by colchicine, although some neurons of paleocortical regions are vulnerable. Colchicine does not appear to be an excitotoxin like kainic acid.The neurotoxicity of colchicine appears to be related to the destruction of microtubules, since other agents which disrupt microtubules have similar toxic effects, and since analogs of colchicine which do not disrupt microtubules are non-toxic. Colchicine may induce an autotoxic response which leads to neuronal death in certain populations due to the accumulation of some toxic cellular product which is normally transported by a microtubule-dependent process. The selective vulnerability of neurons to the neurotoxic effects of colchicine may be a model for system degenerations of the central nervous system in which certain subpopulations of neurons are selectively vulnerable to abnormal accumulations of metabolic products.     

CCK nerve terminals in the rat striatal and limbic areas originate partly in the brain stem and partly in telencephalic structures

After direct injection of colchicine into the rat rostral caudateputamen, levels of cholecystokinin-like immunoreactive (CCK-IR) material in this part of the nucleus are significantly lowered, and CCK-IR neuronal cell bodies are not demonstrable in the caudateputamen, although numerous ones are revealed in some adjacent telencephalic regions. Direct injection of kainic acid into the rostral caudate-putamen is not followed by a decrease of CCK-IR material in the lesioned region. Twenty one days after injection of 6-hydroxydopamine into the rat ventral mesencephalon, a significant decrease of CCK-IR material is observed in the frontal pole, the pyriform cortex, the nucleus accumbens and the ventral mesencephalon itself, but not in the caudate-putamen. After brain stem hemitransection, no decrease in CCK-IR material is observed in the rostral caudate-putamen.     

Comparison of anticonvulsant effect of ethanol against NMDA-, kainic acid- and picrotoxin-induced convulsions in rats

The anticonvulsant effect of ethanol against N-methyl-D-aspartic acid-(NMDA), kainic acid-, and picrotoxin-induced convulsions was studied in rats. Ethanol (2 g/kg, ip) offered protection against these agents, and it was most effective against picrotoxin and least effective against kainic acid. MK801, NMDA receptor antagonist, also provided protection against these agents. However, it was most effective against NMDA and least effective against kainic acid. Ineffective doses of MK801 (0.1 mg/kg, ip) and ethanol (0.5 g/kg, ip), when administered concurrently, had a facilitatory anticonvulsant effect, thereby providing protection against mortality following severe convulsions induced by NMDA or picrotoxin, but not against kainic acid. The protective effect of ethanol against NMDA- and kainic acid-induced neurotoxicity, in contrast to picrotoxin-induced toxicity, was not reversed by imidazodiazepine, Ro 15-4513, an ethanol antagonist. Furthermore, Ro 15-4513 did not produce any proconvulsant effect with NMDA or kainic acid. These findings suggested that the anticonvulsant actions of ethanol may be attributed to its ability to antagonize NMDA-mediated excitatory responses and facilitate the GABAergic transmission.     

Kainic acid-induced seizures: changes in brain extracellular ions as assessed by intracranial microdialysis

The effect of kainic acid on extracellular [K+], [Ca2+], and [Na+] in the rat piriform cortex and hippocampus was studied by means of intracranial microdialysis. Either a dialysis fiber loop or horizontal Vita fiber were stereotaxically implanted within the piriform cortex or hippocampus, respectively. About 24 h later, fibers were perfused (1 ml/min) with Krebs-Ringer bicarbonate solution. Effluent samples were collected before (four at 30 min intervals), and after (six at 30 min intervals) administration of kainic acid (16 mg/kg, i.p.) or kainic acid vehicle. Kainic acid induced sequential signs of lethargy, staring, "wet-dog shakes," forepaw clonus, and tonic-clonic convulsions. In these awake free-moving rats, kainic acid induced a rapid and prolonged increase in extracellular [K+] and an apparent, but not statistically significant, decrease in extracellular [Ca2+] within the hippocampus. In the piriform cortex, kainic acid induced increases in extracellular [K+] and [Na+], which were associated with early pre-convulsive signs. In contrast to the pronounced ion changes commonly seen when the brain is activated by factors such as local application of excitatory substances or when the brain is made ischemic or hypoxic, extracellular ion concentrations are relatively well maintained during parenteral kainic acid-induced seizures.     

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.     

Effects of felbamate,kynurenic acid derivatives and nmda antagonists on in vitro kainate-induced epileptiform activity

The effects of the novel anticonvulsant felbamate, which binds to the 5–7 dichlorokynurenic binding sites, were tested towards the CA1 epileptiform activity induced in rat hippocampal slices by kainic acid. The effects of the kynurenic acid derivatives 7-chlorokynurenic acid and 5–7-dichlorokynurenic acid and of the NMDA antagonists COS 19755, MK-801 and ketamine were also studied for comparison. Slice perfusion with 1 μM kainic acid produced within 30 min the development of an evoked CA1 epileptiform bursting made up by an increase in amplitude of the primary population spikes followed by the appearance of secondary epileptiform population spikes. Slice perfusion with CGS 19755 (100 μM) or MK-801 (100 μM) or ketamine (100 μM) failed to affect within 30 min the CA1 epileptiform activity due to kainic acid. On the contrary, slice perfusion with felbamate (1.3–1.6 mM) or 7-chlorokynurenic acid (100 μM) or 5–7-dichlorokynurenic acid (100 μM) produced within 30 min a significative (P < 0.05) decrease of the kainate-induced epileptiform bursting duration. The results indicate that felbamate and kynurenic acid derivatives but not NMDA antagonists present an inhibitory effect against the epileptiform activity due to kainic acid.     

Metabolic and morphologic effects of colchicine on human T-lymphocyte expression of Fc mu and Fc gamma receptors

The influence of colchicine on human T-cell Fc mu- and Fc gamma-receptor expression during culture was studied utilizing a rosette technique with bovine erythrocytes coated with IgM (EOx-IgM) or IgG (EOx-IgG). Treatment of T cells with greater than or equal to 10(-6) M concentrations of colchicine induced in these cells progressive loss of microtubules and surface microvilli, inhibited their Fc mu-, but not Fc gamma-receptor expression during culture, and increased their cyclic AMP levels. However, similar treatment of cells with lumicolchicine, a photoinactivated isomer, identically inhibited the T-cell Fc mu-receptor expression as well, without inducing loss of microtubules or microvilli or raising cyclic AMP levels in them. A direct influence on T-cell protein synthesis by either colchicine or lumicolchicine is likely, as greater than or equal to 10(-6) M concentrations of alkaloid identically inhibited [3H]leucine incorporation and Fc mu-receptor expression by T cells without inhibiting their alpha-methyl isobutyric acid transport. No impairment of optimal EOx-IgM rosette formation occurred in control T lymphocytes cultured for 24 hr and then treated with colchicine, which suggests that its effects did not directly influence the receptor-ligand interaction itself. These findings suggest colchicine has several sites of action on T cells, dependent and independent of microtubular depolymerization, which may be responsible for alterations of T-lymphocyte cellular metabolism and function.     

CYCLIC NUCLEOTIDE ACCUMULATION IN VITRO IN THE CEREBELLUM OF ''NERVOUS'' NEUROLOGICALLY MUTANT MICE

Abstract— Slices of cerebellum from Purkinje cell-deficient, neurologically mutant 'nervous' mice or normal littermates synthesized cyclic AMP and cyclic GMP during in vitro incubations. Resting levels of cyclic AMP were the same in the two groups, but accumulations in the presence of kainic acid, a glutamic acid analogue, or norepinephrine were significantly greater in the 'nervous' mice. Resting levels of cyclic GMP were lower in the 'nervous' mice, but the elevations produced by kainic acid were the same in both groups. Adenylate and guanylate cyclase activities in the cerebellum were not affected by the mutation. These findings indicate that cyclic nucleotide synthesis in the cerebellum does not occur solely in the Purkinje cell population.     

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.     

The long-term memory retrieval following kainic acid administration

Effect of the neurotoxin kainic acid to the food-procuring task were studied in Wistar rats. A single injection of the acid in subconvulsive dose (8 mg/kg) impaired the task performance within some weeks but not immediately after the treatment. Higher doses of kainic acid (10 mg/kg) impaired the task performance within a few hours after treatment for up to 10 days. The treatment did not prevent rat's learning of a new task in the same experimental chamber. The revealed deficit in the long-term memory retrieval might be explained by specific effects of kainic acid upon the hippocampal system.     

Choline Acetyltransferase Activity in the Rat Brain Cortex Homogenate, Synaptosomes, and Capillaries After Lesioning the Nucleus Basalis Magnocellularis

Stereotaxic lesions of the nucleus basalis magnocellularis were made unilaterally in male Wistar rats with either kainic or ibotenic acid, using the contralateral side as control. Differences in behavior, body weight, and survival were observed between the kainic and ibotenic acid-treated rats. One week after surgery, the rats were sacrificed and the effect of the lesions on choline acetyltransferase activity was measured in brain cortex homogenate, synaptosomes, and capillaries. In kainic acid-lesioned rats, choline acetyltransferase activity decreased in homogenate and synaptosomes of the ipsilateral side with respect to that of the contralateral side; but the ibotenic acid lesion, which also reduced the ipsilateral choline acetyltransferase activity in homogenate, showed a rather different effect on the enzymatic activity of the synaptosomes. There were also differences between the effect of kainic and ibotenic acid lesions on choline acetyltransferase activity in the capillaries of the ipsilateral side with respect to that of the contralateral one. However, capillary choline acetyltransferase activity of the treated rats was in both sides three times higher than that of unoperated rats.     

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