Microglia activation contributes to quinolinic acid-induced neuronal excitotoxicity through TNF-α.

It has been reported that activation of NF-κB is involved in excitotoxicity; however, it is not fully understood how NF-κB contributes to excitotoxicity. The aim of this study is to investigate if NF-κB contributes to quinolinic acid (QA)-mediated excitotoxicity through activation of microglia. In the cultured primary cortical neurons and microglia BV-2 cells, the effects of QA on cell survival, NF-κB expression and cytokines production were investigated. The effects of BV-2-conditioned medium (BCM) on primary cortical neurons were examined. The effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, and minocycline (MC), an inhibitor of microglia activation, on QA-induced excitotoxicity were assessed. QA-induced NF-κB activation and TNF-α secretion, and the roles of TNF-α in excitotoxicity were studied. QA at the concentration below 1 mM had no apparent toxic effects on cultured primary neurons or BV-2 cells. However, addition of QA-primed BCM to primary neurons did aggravate QA-induced excitotoxicity. The exacerbation of QA-induced excitotoxicity by BCM was partially ameliorated by inhibiting NF-κB and microglia activation. QA induced activation of NF-κB and upregulation of TNF-α in BV-2 cells. Addition of recombinant TNF-α mimicked QA-induced excitotoxic effects on neurons, and neutralizing TNF-α with specific antibodies partially abolished exacerbation of QA-induced excitotoxicity by BCM. These studies suggested that QA activated microglia and upregulated TNF-α through NF-κB pathway in microglia. The microglia-mediated inflammatory pathway contributed, at least in part, to QA-induced excitotoxicity.

The calcium channel blocker nifedipine attenuates slow excitatory amino acid neurotoxicity.

High concentrations of potent N-methyl-D-aspartate (NMDA) agonists can trigger degeneration of cultured mouse cortical neurons after an exposure of only a few minutes; in contrast, selective non-NMDA agonists or low levels of NMDA agonists require exposures of several hours to induce comparable damage. The dihydropyridine calcium channel antagonist nifedipine was used to test whether this slow neurotoxicity is mediated by a calcium influx through voltage-gated channels. Nifedipine had little effect on the widespread neuronal degeneration induced by brief exposure to high concentrations of NMDA but substantially attenuated the neurotoxicity produced by 24-hour exposure to submaximal concentrations of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate, kainate, or quinolinate. Calcium ion influx through dihydropyridine-sensitive, voltage-dependent calcium channels may be an important step in the neuronal injury induced by the prolonged activation of NMDA or non-NMDA glutamate receptors.

On the excitotoxic properties of quinolinic acid, 2,3-piperidine dicarboxylic acids and structurally related compounds.

To obtain information about the receptors which mediate the neurotoxic actions of quinolinic acid, a series of pyridine dicarboxylates and piperidine dicarboxylates and structurally related compounds were tested for their neurotoxic effects following intrastriatal or intrahippocampal infusion in the rat, and for their activity in assays of binding and uptake sites for acidic amino acids. Of the compounds tested, only cis- and trans-2,3-piperidine dicarboxylates and quinolinic acid showed pronounced neurotoxic effects. At 600 nmol, 2,6- and 3,4-pyridine dicarboxylates were weakly active and the remaining compounds were inactive in both brain regions. After injection into the striatum of the adult rat, trans-2,3-piperidine dicarboxylate, quinolinic acid and cis-2,3-piperidine dicarboxylate caused axon-sparing neuronal degeneration as assessed by light microscopic and neurochemical methods, the threshold doses being 12, 24 and 120 nmol, respectively. In the striatum of the 7-day old rat, 30 nmol quinolinic acid or 600 nmol cis-2,3-piperidine dicarboxylate were inactive. Small doses of cis-2,3-piperidine dicarboxylate (60 nmol) and quinolinic acid (30 nmol) injected into the adult rat hippocampus resulted in a preferential loss of pyramidal neurons. In larger doses granule cells also degenerated. In contrast, trans-2,3-piperidine dicarboxylate was equally toxic to hippocampal neurons, regardless of the dose used. No "distant" neuronal damage was observed after the intracerebral application of any test compound. Equimolar amounts of (-)-2-amino-7-phosphonoheptanoic acid completely blocked the neurotoxic effects of quinolinic acid, cis- and trans-2,3-piperidine dicarboxylate after injection into the striatum or hippocampus. None of the analogs tested were good inhibitors of Cl--dependent or independent binding of L-[3H]glutamate, [3H]kainate or high-affinity, Na+-dependent uptake of L-glutamate in striatal or hippocampal tissue at 1 mM. The results indicate that the receptors mediating the neurotoxic effects of these compounds have strict structural requirements for activation. Whereas the excitotoxic characteristics of trans-2,3-piperidine dicarboxylate suggest a direct action on N-methyl-D-aspartate receptors, the properties of quinolinic acid and cis-2,3-piperidine dicarboxylate are far more complex and make categorization of their receptor-interactions difficult. Indirect mechanisms may account for the excitotoxicity of quinolinic acid and cis-2,3-piperidine dicarboxylate.

The basolateral amygdala-ventral striatal system and conditioned place preference: further evidence of limbic-striatal interactions underlying reward-related processes.

The effects on the expression of a conditioned place preference of bilateral, excitotoxic amino acid-induced lesions of the basolateral region of the amygdala, or the ventral striatum, or asymmetric, unilateral lesions of both structures were studied. The place preference was conditioned by exposing hungry rats to sucrose in a distinctive environment. Following acquisition, bilateral quisqualate-induced lesions of the basolateral amygdala, as well as bilateral quinolinate-induced lesions of the ventral striatum, abolished the conditioned place preference. Bilateral ventromedial, but not dorsolateral, quinolinate-induced caudate-putamen lesions attenuated the place preference. Combining a unilateral lesion of the basolateral amygdala with a contralateral lesion of the ventral striatum also disrupted the conditioned place preference. These data provide further support for the hypothesis that the basolateral amygdala and ventral striatum are important parts of a neural system subserving stimulus-reward associations.

Quinolinate neurotoxicity and glutamatergic structures.

Neurotoxic properties of quinolinic acid following intracerebroventricular application were investigated in the hippocampal formation of 12- and 30-day-old rats. Quinolinic acid neurodegenerative potency was found to depend on the survival time, the dose applied and the developmental stage of the animal. Pretreatment with kynurenic acid and ketamine as well as the transection of the perforant path were noted to protect major parts of the hippocampal cell layers from quinolinic acid-induced degenerative effects. The results are interpreted in view of a putative dependence of quinolinic acid neurotoxicity on the presence of established synaptic, in particular glutamatergic, processes which play a major role in the hippocampal formation and mature during the first postnatal weeks. For comparison, we studied local effects of quinolinic acid on superior cervical and dorsal root ganglia in which glutamate inputs obviously do not occur; no signs of neuronal vulnerability were seen.

Adenosine A2 receptors: selective localization in the human basal ganglia and alterations with disease.

Adenosine A2 receptors were labeled and visualized by autoradiography in tissue sections of the human brain using the A2-selective agonist ligand [3H](2-p-(2-carboxyethyl)phenylamino)-5'-N-carboxamidoadenosine (CGS 21680). The binding of this ligand was of high affinity, reversible, and was blocked by adenosine A2 agents. Autoradiographic mapping of adenosine A2 sites revealed them to be exclusively restricted to the caudate nucleus, putamen, nucleus accumbens, olfactory tubercle and the lateral segment of the globus pallidus. The densities of adenosine A2 receptors in other brain areas did not differ from background levels. This selective localization prompted us to study the consequences of neurodegenerative diseases such as Parkinson's disease and Huntington's chorea on the densities and localization of these sites in the basal ganglia. In Parkinson's disease the density of adenosine A2 binding sites was comparable to that seen in control cases. In contrast, density values of A2 sites were dramatically decreased, compared to control values, in the basal ganglia of patients with Huntington's chorea. Similar losses of A2 receptors were observed in the guinea-pig striatum after local application of quinolinic acid while lesioning of the dopaminergic neurons was without effect. All these results taken together suggest that adenosine A2 receptors are localized on striatal output neurons which degenerate in Huntington's chorea.

Functional and histological consequences of quinolinic and kainic acid-induced seizures on hippocampal somatostatin neurons.

Changes in endogenous somatostatin after quinolinic and kainic acids were investigated by measuring somatostatin-like peaks by in vivo voltammetry and by assessing the distribution of somatostatin-positive neurons by immunocytochemistry. Kainic acid (0.19 nmol/0.5 microliter) or quinolinic acid (120 nmol/0.5 microliter) in doses inducing comparable electroencephalographic seizure patterns, were injected into the hippocampus of freely moving rats. Somatostatin-like peaks were measured every 6 min for 3 h by a carbon fiber electrode implanted in the proximity of the injection needle. Kainic acid kept somatostatin-like peaks significantly higher than saline from 48 min after the injection till the end of the recording. Somatostatin-like peaks were dramatically elevated by quinolinic acid, reaching a maximum of 482% 60 min after the injection. Three days later, administration of kainic acid resulted in selective degeneration of CA3 pyramidal neurons but did not affect the number of somatostatin-positive cells, while quinolinic acid induced cell loss in all pyramidal layers and complete degeneration of somatostatin-positive cells in the whole hippocampus. Thus, the quantitative difference in somatostatin release in response to doses of kainic and quinolinic acids inducing comparable electroencephalographic seizure patterns was reflected in a substantial difference in the neurodegenerative consequences. In both models, the release of somatostatin in response to seizures may be interpreted as a "defense" mechanism aimed at reducing the spread of excitation in the tissue.

Loss of the acoustic startle response following neurotoxic lesions of the caudal pontine reticular formation: possible role of giant neurons.

The effect of the excitotoxic N-methyl-D-aspartate agonist quinolinic acid in the caudal pontine reticular formation on the acoustic startle response was investigated in rats. Bilateral injections of 90 nmol of quinolinic acid led to large lesions in the reticular formation characterized by the loss of all neurons and a marked reduction or even abolition of the acoustic startle response; 18 nmol of quinolinic acid led to smaller lesions characterized by a selective loss of giant neurons within the caudal pontine reticular formation and a reduction of the startle amplitude. The partial correlation analysis revealed that the reduction of the amplitude of the acoustic startle response can be correlated with the loss of the giant neurons (r = 0.575; d.f. = 29; P less than 0.001) but not with the reduction of the number of all neurons (r = 0.207; d.f. = 29; P greater than 0.2) in the caudal pontine reticular formation. These findings were reconciled with electrophysiological and anatomical data indicating that the giant neurons in the caudal pontine reticular formation receive acoustic input and project to motoneurons of the spinal cord. It is concluded that the caudal pontine reticular formation is an important element of the startle pathway and that the giant reticulospinal neurons constitute an important part of the sensorimotor interface mediating this response.

The basolateral amygdala regulates sensorimotor gating of acoustic startle in the rat.

The acoustic startle reflex is a coordinated contraction of the skeletal musculature in response to a sudden, intense sound. One form of startle plasticity, "prepulse inhibition", is the normal suppression of the startle reflex when the intense startling stimulus is immediately preceded by a weak pre-stimulus. Prepulse inhibition is utilized as an operational measure of sensorimotor gating, and is significantly impaired in several neuropsychiatric disorders that are characterized by symptoms associated with central inhibitory deficits. In rats, prepulse inhibition is disrupted by central dopamine activation or by manipulations of limbic cortical structures including the prefrontal cortex and hippocampus. In the present study, we assessed prepulse inhibition in rats after surgical and pharmacologic manipulations of the basolateral amygdala. Quinolinic acid lesions of the basolateral amygdala significantly reduced prepulse inhibition without significantly changing startle amplitude. These lesions also blocked fear-potentiated startle, which is known to be regulated by the basolateral amygdala. The prepulse inhibition-disruptive effects of basolateral amygdala lesions were not reversed by systemic injection of the dopamine antagonist haloperidol at doses that totally restored prepulse inhibition in apomorphine-treated rats. In other studies, intra-amygdala infusion of the competitive N-methyl-D-aspartate antagonist DL-2-amino-5-phosphonovaleric acid (0, 0.15, 1.5, 4.5 microg) dose-dependently reduced prepulse inhibition. These data suggest that the basolateral amygdala regulates sensorimotor gating by mechanisms that are independent of central dopamine hyperactivity.

Prevention of quinolinic acid neurotoxicity in rat hippocampus in vitro by zinc. Ultrastructural observations.

The effect of zinc on the development and evolution of quinolinic acid-induced alterations in the rat hippocampus in culture was studied ultrastructurally. Zinc, although it possesses intrinsic cytotoxic properties, after application in concentrations comparable with those encountered in vivo, was able to prevent typically observed responses after quinolinic acid exposure, either early or late damage to hippocampal neurons. The results further support the concept of a potential protective effect of zinc against the neurotoxicity of particular excitotoxins.

Quinolinate neurotoxicity in cortical cell culture.

The central neurotoxicity of the endogenous tryptophan metabolite, quinolinate, has been postulated to participate in the pathogenesis of the neuronal cell loss associated with several neurological disease states. In the present study, quinolinate neurotoxicity was quantitatively studied in dissociated cell cultures prepared from the fetal mouse neocortex. Sufficient exposure of cortical cultures to quinolinate was associated with considerable neuronal cell loss, but no glial cell loss; this neurotoxicity could be blocked by 2-amino-5-phosphonovalerate and kynurenate, drugs known to block N-methyl-D-aspartate receptors. The quinolinate dose-toxicity relationship showed that the potency of quinolinate as a neurotoxin is relatively low, especially with brief (20 min) exposure times, where an ED50 of 2 mM was observed. However, with longer exposure times of 24 and 96 h, quinolinate is more potent: the latter exposure was characterized by an ED50 of 250-400 microM. Ion substitution experiments suggested that quinolinate neurotoxicity can be separated into two distinct components on the basis of differences in time course and ionic dependence: an acute, sodium-dependent "excitotoxic" component, marked by early cell swelling; and a late, calcium-dependent component, marked by delayed cell degeneration. Acute neuronal swelling was seen only with exposure to quinolinate concentrations in excess of 1 mM, so under actual pathophysiological conditions, quinolinate neurotoxicity might be nearly completely related to the calcium-dependent component, with little or no "excitotoxic" contribution.

An investigation of the mechanisms of delayed neurodegeneration caused by direct injection of quinolinate into the rat striatum in vivo.

Injection of the N-methyl-D-aspartate receptor agonist quinolinate, or N-methyl-D-aspartate itself, into the rat brain produces neurodegeneration which can be prevented by N-methyl-D-aspartate receptor antagonists administered up to 5 h after excitotoxin injection. The present study was designed to investigate aspects of the mechanisms involved in this delayed form of neurodegeneration. Following its injection into the rat striatum, extracellular levels of [3H]quinolinate were monitored using a microdialysis probe located 1 mm from the site of injection. Peak concentrations were observed 10-20 min after injection and [3H]quinolinate levels decayed in a biexponential fashion, the initial component having an apparent t1/2 of 13.7 +/- 5.2 min (n = 3). Estimations of the extracellular concentrations of quinolinate after an injection of 200 nmol indicated a peak level of 13.7 +/- 6.0 mM (n = 3) at 10-20 min which declined to 1.2 +/- 0.13 mM (n = 3) by 2 h and substantial levels were present up to 5 h, the period over which N-methyl-D-aspartate receptor antagonists are effective in this model. Administration of dizocilpine at 1, 2, 3 or 5 h after injection of 100, 200 or 400 nmol quinolinate resulted in a similar temporal profile of neuroprotection, as assessed by measuring the activities of choline acetyltransferase and glutamate decarboxylase in striatal homogenates, which was independent of the degree of neurodegeneration produced by the different excitotoxin doses. Overall, these results suggest that the neuronal degeneration caused by quinolinate in vivo is critically dependent upon events occurring after the initial peak of excitoxin levels in the extracellular space.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of alpha-phenyl-tert-butyl nitrone on neuronal survival and motor function following intrastriatal injections of quinolinate or 3-nitropropionic acid.

We have investigated the neuroprotective effects of the the spin-trapping agent alpha-phenyl-tert-butyl nitrone on striatal lesions produced by local injections of the excitotoxin quinolinate or the mitochondrial toxin 3-nitropropionic acid. We have assessed both the behavioural and morphological consequences of the lesion. Thus, we tested paw-reaching ability and amphetamine- and apomorphine-induced rotational behaviour in lesioned rats with or without alpha-phenyl-tert-butyl nitrone treatment, and also explored the relationship between the outcome of the behavioural studies and the extent of the lesion. In the morphological analysis, we chose immunocytochemistry for dopamine- and cyclic AMP-regulated phosphoprotein with a molecular weight of 32,000 as a specific marker for striatal neurons. The paw-reaching ability of rats with the quinolinate and 3-nitropropionic acid lesions was significantly impaired compared to normal control animals. Treatment with alpha-phenyl-tert-butyl nitrone significantly ameliorated the paw-reaching deficits produced by the quinolinate lesion, whereas the 3-nitropropionic acid-induced deficits were unaffected by alpha-phenyl-tert-butyl nitrone. Both quinolinate and 3-nitropropionic acid lesions resulted in a rotation asymmetry towards the lesioned side in response to both amphetamine and apomorphine. In the quinolinate lesion model, the alpha-phenyl-tert-butyl nitrone treatment resulted in a less marked motor asymmetry in response to both drugs. By contrast, alpha-phenyl-tert-butyl nitrone did not significantly reduce the drug-induced rotation asymmetry in rats with a 3-nitropropionic acid lesion. Morphological analyses disclosed that alpha-phenyl-tert-butyl nitrone significantly increased the size of the spared striatum in the quinolinate lesions, but only caused a non-significant trend towards an attenuation of the 3-nitropropionic acid lesions. The behavioural deficits were inversely correlated to the size of the spared residual striatum. The intrastriatal injection of 3-nitropropionic acid, unlike the injection of quinolinate, caused degeneration of the nigrostriatal dopamine system as well as of transverse fibre bundles of the internal capsule in the striatum, in addition to the striatal lesion. The behavioural studies revealed that the combination of multiple lesions seen in 3-nitropropionic acid-lesioned rats significantly exacerbated paw-reaching deficits and amphetamine-induced rotation asymmetry. In conclusion, alpha-phenyl-tert-butyl nitrone attenuated behavioural and morphological consequences of striatal lesions induced by local injections of quinolinate, but not of 3-nitropropionic acid. Deficits in behavioural tests of striatal function reflected well the extent of striatal lesion. The intrastriatal injection of 3-nitropropionic acid led to degeneration of both intrinsic striatal neurons and the nigrostriatal dopaminergic system, suggesting that this lesion may provide an animal model of a form of multiple system atrophy rather than Huntington's disease.

Intracerebral implantation of nerve growth factor-producing fibroblasts protects striatum against neurotoxic levels of excitatory amino acids.

With the exception of L-DOPA pharmacological treatment in Parkinson's disease, the neurodegenerative diseases lack effective treatment. Previous studies of neurodegenerative diseases suggest that symptoms arise secondary to defects in local neuronal circuitry and cannot be treated effectively with systemic drug delivery. Therefore, a promising treatment is the application of fetal or genetically engineering cells which protect or replace neurons in deficient regions. Engineered cells can be derived from cell lines or grown from recipient host fibroblasts or other cells, then modified to produce and secrete substances at a specific area of the brain. A previous study using parallel intracerebral infusions of nerve growth factor and an excitotoxic amino acid into the rat striatum demonstrated a protective effect of nerve growth factor on neurons [Aloe L. (1987) Biotechnology 5, 1085-1086]. In order to further test this paradigm, we have utilized a biological delivery system of nerve growth factor by implanting fibroblasts into the rat striatum which secrete high levels of nerve growth factor, prior to infusing the neurotoxins quinolinate or quisqualate. Animals in this group had smaller lesions than did a group implanted with a similar non-nerve growth factor-producing graft. In addition, marked neuronal sparing was noted within areas of lesions in those animals containing a nerve growth factor-producing graft. These results indicate that implantation of genetically engineered nerve growth factor-secreting cells can be used to protect neurons at a specific target from excitotoxin-induced lesions.

Calbindin-D28K-containing neurons in animal models of neurodegeneration: possible protection from excitotoxicity.

Brain levels of the calcium binding protein Calbindin-D28K (CaBP28K) and CaBP28K mRNA were measured for various animal models of neurodegenerative diseases (MPTP-treated C57BL/6J mice and Sprague-Dawley rats receiving striatal/intraperitoneal kainic acid or quinolinic acid into the nucleus basalis magnocellularis). Brain areas were tested (radioimmunoassay, Western blot, slot blot, and Northern blot) for levels of CaBP28K and CaBP28K mRNA. The various models did not exhibit any changes in protein or mRNA levels from the controls, suggesting that CaBP28K-containing neurons were not lost after exposure to these neurotoxins. Immunocytochemical characterization of the substantia nigra of the MPTP-treated mice revealed that there was significant dopaminergic cell loss in this brain area after MPTP treatment. The majority of dopaminergic neurons that degenerated did not contain CaBP28K. The small percentage of surviving neurons were CaBP28K-positive. These results suggest that the presence of CaBP28K may protect neurons from calcium-mediated neurotoxicity.

Nerve cell death induced in vivo by kainic acid and quinolinic acid does not involve apoptosis.

We investigated whether in vivo excitotoxicity was mediated by a mechanism of programmed cell death called apoptosis. Neurotoxic doses of kainic acid (1.2 nmol) and quinolinic acid (120 nmol) were unilaterally injected in the dorsal hippocampus of anesthetized rats. Eight or 16 h later the animals were killed and DNA was extracted from the injected hippocampi. DNA from mouse thymocytes exposed to methylprednisolone (10(-5) M for 6 h at 37 degrees C) was used as a positive control of apoptotic cells. No typical 'ladder' of DNA fragments (multimers of approximately 200 Kb) which characterizes apoptosis was seen in hippocampal cells after toxic doses of kainic or quinolinic acid, as assessed by agarose gel electrophoresis. This suggests that hippocampal nerve cell death induced in vivo by the excitotoxins is not mediated by apoptosis.

A novel device for chronic intracranial drug delivery via microdialysis.

A system is described for chronic intracranial drug administration in the rat using a modified in vivo microdialysis probe coupled to an Alzet model 2002 osmotic minipump. The results presented demonstrate that this system can be used for the chronic administration of quinolinic acid with minimal non-specific damage. Each pump delivered approximately 225 microliters of solution over a period of 19-20 days when tested in vitro. The dialysis units were uniform in function, delivering greater than 93% of the [3H]quinolinic acid initially loaded into the minipump. For in vivo analysis of this apparatus the dose of quinolinic acid tested produced extensive destruction of the striatum. The present system allows reliable drug diffusion over a relatively large area without pressure injection variability. In conclusion, we have developed a simple and inexpensive technique for administration of drugs into brain parenchyma with substantial advantages over previously used techniques.

Quinolinic acid concentrations in striatal extracellular fluid reach potentially neurotoxic levels following systemic L-tryptophan loading.

Following a systemic tryptophan load, striatal extracellular fluid levels of quinolinic acid in the rat were quantified using intracerebral microdialysis. After an intraperitoneal dose of L-tryptophan (250 mg/kg), quinolinic acid levels in striatal perfusates increased by 230 fold. Peak concentrations of quinolinic acid exceeded 10(-5)M, a concentration previously shown to be neurotoxic in vitro. These results indicate that quinolinic acid is markedly precursor responsive and that its concentration in striatal extracellular fluid may reach neurotoxic levels following an acute tryptophan load.