Characterization of temperature rise of the brain and the rectum following intracerebroventricular administration of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and kainate in rats

Intracerebroventricular administration of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) or kainate caused a rise of the temperature of the brain and the rectum in urethane-anesthetized rats. An AMPA–kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), significantly suppressed the AMPA- and kainate-induced rises of brain and rectal temperatures. An N-methyl- -aspartate receptor antagonist, MK-801, also suppressed the rises of the brain and rectal temperatures induced by AMPA or kainate, but the profiles of the suppressive effects of MK-801 were different between rats treated with AMPA and kainate. An antipyretic agent, indomethacin, completely suppressed the AMPA-induced rises of brain and rectal temperatures. Although indomethacin completely suppressed the kainate-induced rise of the rectal temperature as well, the brain temperature was still raised. These findings suggest that distinct mechanisms may be involved in the temperature rise of the brain and the rectum mediated through AMPA and kainate receptor stimulation.     

AMPA receptor-mediated slow neuronal death in the rat spinal cord induced by long-term blockade of glutamate transporters with THA

Excitotoxicity secondary to the loss of glutamate transporters (GluT) has been proposed as a possible pathogenetic mechanism for neuronal degeneration in amyotrophic lateral sclerosis. We therefore investigated whether prolonged in vivo pharmacologic inhibition of GluT would result in neuronal damage in the rat.

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-Threo-β-hydroxyaspartate (THA), a potent GluT inhibitor, and glutamate were continuously infused into the rat spinal subarachnoid space by using a mini-osmotic pump. Animals that received both THA and glutamate, but not those received either singly, displayed tail paralysis with or without hind-limb paralysis and urinary incontinence after the third postoperative day. Pathologically, symptomatic animals exhibited neuronal loss with a variable extent of gliosis preferentially involving the dorsal horn of the lumbosacral cord. In the rostral spinal segments adjacent to those regions of intense pathologic changes, small neurons in the dorsal horn were selectively destroyed, a pattern similar to the late-onset neuronal damage induced by continuous intrathecal administration of 1-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) [R. Nakamura et al., Brain Res. 654 (1994) 279–285]. These behavioral and pathologic changes were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), suggesting that pharmacologic blockade of GluT causes selective neuronal damage in vivo by AMPA receptor activation.     

Cellular expression of ionotropic glutamate receptor subunits in subpopulations of neurons in the rat substantia nigra pars reticulata

In order to characterize the expression of ionotropic glutamate receptor immunoreactivity in subpopulations of neurons in the rat substantia nigra pars reticulata (SNr), double labeling experiments were performed. Neurons in the reticulata were found to display GluR1, GluR2, GluR2/3, GluR4, N-methyl-D-aspartate receptor 1 (NMDAR1) and NMDAR2B immunoreactivity. Some of the reticulata neurons were shown to display GluR1 and GluR2 immunoreactivity or GluR2 and GluR4 immunoreactivity at the single cell level. In addition, subpopulations of reticulata neurons were characterized on the basis of the strong expression of parvalbumin (PV) and GABA transaminase immunoreactivity. All of the reticulata neurons that displayed strong immunoreactivity for PV or GABA transaminase also displayed immunoreactivity for GluR1, GluR2/3, GluR4, NMDAR1 and NMDAR2B. A tiny portion (around 15%) of reticulata neurons that display NMDAR1 immunoreactivity was found to be PV- or GABA-transaminase-negative. The present results indicate that native alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type receptors and NMDA-type receptors in the rat substantia nigra are composed of heteromeric receptor subunits. The present findings further demonstrate that most of the AMPA-type and NMDA-type glutamate receptor subunits are primarily expressed by subpopulations of neurons in the rat SNr.     

Free radicals and lipid peroxidation do not mediate β-amyloid-induced neuronal cell death

“β Amyloid (Aβ)-induced free radical-mediated neurotoxicity” is a leading hypothesis as a cause of Alzheimer's disease (AD). Aβ increased free radical production and lipid peroxidation in PC12 nerve cells, leading to increased 4-hydroxy-2-nonenal (HNE) production and modification of specific mitochondrial target proteins, apoptosis and cell death. Pretreatment of the cells with isolated ginkgolides, the anti-oxidant component of Ginkgo biloba leaves, or vitamin E, prevented the Aβ-induced increase of reactive oxygen species (ROS). Ginkgolides, but not vitamin E, inhibited the Aβ-induced HNE modification of mitochondrial proteins. However, treatment with these anti-oxidants did not rescue the cells from Aβ-induced apoptosis and cell death. These results indicate that free radicals and lipid peroxidation may not mediate Aβ-induced neurotoxicity.     

姜黄素对AMPA/KA受体介导大鼠海马神经元钙内流的影响

目的 探讨姜黄素对α-氨基-3-羧基-5-甲基异恶唑-4-丙酸(AMPA)/海人酸(KA)受体介导大鼠海马神经元钙内流的影响.方法 选用胚胎17dSD鼠分离海马,离体培养海马神经元,借助活体钙荧光染色和激光共聚焦钙成像技术观察100μmol/LKA刺激海马神经元内钙的变化,不同浓度(5、10、15、30、50 μmol/L)姜黄素预孵育海马神经元30min对100μmol/L KA刺激下细胞内钙变化的影响,15 μmol/L姜黄素对不同浓度(10、30、50、100、200、300 μmol/L)KA刺激海马神经元内钙变化的影响.应用钴染色技术观察(30、100 μmol/L KA)刺激后海马神经元钴阳性染色细胞变化.姜黄素预孵育30min对KA刺激导致钴阳性染色细胞变化的影响.结果 不同浓度姜黄素预孵育30 min均可以明显缓解100 μmol/L或30 μmol/L KA导致的细胞内钙升高程度.差异均有统计学意义(P<0.05),其中15 μmol/L姜黄素作用最为明显.30μmol/L或100 μmol/LKA刺激均可以引起海马神经元钴染色阳性细胞增加,15 μmol/L姜黄素预处理30 min后明显减少钴染色阳性细胞,差异有统计学意义(P<0.05),而其他浓度(5 μmol/L或30 μmol/L)姜黄素未见明显影响.结论 一定浓度的姜黄素可以影响AMPA/KA受体介导大鼠海马神经元钙内流.这可能是姜黄素抗癫痫作用的一个机制.     

Dual effects of interleukin-1β on N-methyl- -aspartate-induced retinal neuronal death in rat eyes

In this study we determine if interleukin-1β (IL-1β) modulates N-methyl- -aspartate (NMDA)-induced retinal damage. Sprague–Dawley rats were anesthetized with inhalation of halothane, after which a single injection of 5 μl of IL-1β (0.1 to 10 ng/eye) (and/or IL-1 receptor antagonist (IL-1ra)) for experimental eyes was administered. Two days later (or simultaneously), NMDA (20 nmol) was injected into the vitreous space. One week later, each eye was enucleated and transverse sections were subjected to morphometric analysis. Enzyme-linked immunosorbent assay (ELISA) was conducted for the determination of IL-1β levels in retina. Immunohistochemical and immunoblot studies were also performed. In eyes that received an intravitreal injection of IL-1β (0.1 to 10 ng/eye), significant thinning of the inner plexiform layer (IPL) was observed (P<0.05). Immunohistochemical and ELISA studies demonstrated upregulated expression of IL-1β in retinas that had undergone NMDA injection. Treatment with 10 ng of IL-1ra induced a protective effect against NMDA-induced retinal damage. Pretreatment with IL-1β induced a significant protective effect on NMDA-induced retinal damage. Our studies suggest that IL-1β induces neuronal cell death directly, as shown by the protective effects of IL-1ra, but has a protective effect on NMDA-induced retinal damage indirectly after an incubation time of at least 2 days.     

Long-term efficacy of low-dose perampanel for progressive myoclonus epilepsy in a patient with Gaucher disease type 3

PurposeWe report the case of a patient with progressive myoclonus epilepsy due to Gaucher disease type 3 whose seizures and ability to perform activities of daily living were significantly improved after starting low-dose perampanel therapy.CaseOur patient’s generalized tonic–clonic seizures and myoclonus did not improve despite the administration of multiple antiseizure medications and enzyme replacement therapy. The myoclonus reduced following pharmacological chaperone therapy, but this effect was temporary, and the generalized tonic–clonic seizures continued to occur. However, the generalized tonic–clonic seizures disappeared following treatment with 2 mg/day of perampanel. In addition, the decrease in myoclonus dramatically improved motor function such as talking, eating, and walking and stabilized the patient’s mental status. These effects have been sustained for more than 4 years.ConclusionPerampanel is expected to be effective in the treatment of progressive myoclonus epilepsy associated with Gaucher disease type 3 and should be considered the drug of choice for this condition.     

Selective changes in AMPA receptors in rabbit cerebellum following classical conditioning of the eyelid-nictitating membrane response

α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors are critically involved in several forms of synaptic plasticity proposed to be neural substrates for learning and memory, e.g., long-term potentiation and long-term depression (LTD). The present study was designed to determine changes in cerebellar AMPA receptors following classical conditioning of the eyeblink-nictitating membrane response (NMR) in the rabbit. Quantitative autoradiography was used to assess changes in ligand binding properties of cerebellar AMPA receptors following NMR conditioning elicited by pairing electrical stimulation of the pontine nuclei with an airpuff to the eye. [³H]AMPA and [³H]-6-cyano-7-nitroquinoxaline-2,3-dion (CNQX) binding were determined following preincubation of frozen–thawed brain tissue sections at 0 or 35°C. With 0°C preincubation, no significant differences in [³H]AMPA binding to cerebellar AMPA receptors were seen between any of the experimental groups tested. In contrast, preincubation at 35°C revealed significant decreases in [³H]AMPA binding to the trained side of the cerebellar cortex resulting from paired presentations of the conditioned and the unconditioned stimuli, while unpaired presentations of the stimuli resulted in no significant effect. With 35°C preincubation, there were no significant differences in [³H]CNQX binding between any of the experimental groups and no significant differences in [³H]AMPA binding in the untrained side of the cerebellum. These results indicate that NMR conditioning is associated with a selective modification of AMPA-receptor properties in brain structures involved in the storage of the associative memory. Furthermore, they support the hypothesis that cerebellar LTD, resulting from decreased synaptic efficacy at parallel fiber–Purkinje cell synapses mediated by a change in AMPA-receptor properties, is a form of synaptic plasticity that supports this type of learning.     

Enhancement of anti-absence effects of ethosuximide by low doses of a noncompetitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist in a genetic animal model of absence epilepsy

N-Acetyl-1-(4-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (THIQ-10c) is a noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist that has been demonstrated to antagonize generalized tonic–clonic seizures in different animal models of epilepsy. In the study described here, we tested the potential effect of such a compound alone or co-administered with ethosuximide in a genetic animal model of absence epilepsy, the WAG/Rij rat. The intraperitoneal or intracerebroventricular microinjection of THIQ-10c alone was unable to significantly modify the number and duration of spike-and-wave discharges (SWDs). In contrast, intracerebroventricular administration of AMPA induced a dose-dependent increase in the number of SWDs. THIQ-10c dose-dependently antagonized this effect. Furthermore, co-administration of THIQ-1c with ethosuximide (50 mg/kg, intraperitoneally) was able to significantly increase the efficacy of the anti-absence drug. In conclusion, although noncompetitive AMPA receptor antagonists alone might not be useful in the treatment of absence epilepsy because of their low therapeutic index, combining them with ethosuximide might be helpful in controlling absence seizures in patients not tolerating this drug or in refractory patients.     

The AMPA antagonist NBQX protects thalamic reticular neurons from degeneration following cardiac arrest in rats

Thalamic reticular (RT) neurons are selectively vulnerable to degeneration following global ischemia. The degenerative mechanism is thought to involve an excitotoxic component, mediated in part by sustained post-ischemia activation of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) type excitatory amino acid (EAA) receptors. In order to test this hypothesis, the selective competitive AMPA type EAA antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxalinedione) was administered at 30 mg/kg to rats 1, 3, and 6 h after resuscitation from 10 min cardiac arrest. NBQX treatment resulted in a 2-fold increase of spared RT neurons, from a mean density of 3.6 ± 0.8 × 10³ neurons/mm³ in cardiac arrest cases to 7.4 ± 1.1 × 10³ neurons/mm³ in the NBQX treated group, which represents sparing of 41.7% of the normal population of RT neurons, and protection of 26.9% of vulnerable RT neurons. Neurons within the central core of the RT manifest both a higher degree of vulnerability to ischemic degeneration, >92% loss, and a higher sensitivity to sparing following NBQX administration, 460% increased sparing, than neuronal sub-populations in the medial or lateral13 of the RT. Protection by post-arrest administration of NBQX suggests that sustained post-arrest stimulation of AMPA receptors is an important component in the process of ischemic degeneration of RT neurons.     

Prevention of HIV-1 gp120-induced neuronal damage in the central nervous system of transgenic mice by the NMDA receptor antagonist memantine

To investigate the in vivo role of NMDA receptor stimulation in HIV-1-related CNS neurotoxicity, we evaluated the neuroprotective potential of the NMDA receptor antagonist memantine in transgenic mice which have gp120-induced CNS damage. Brains of mice treated chronically with memantine and of untreated controls were analysed for structural damage by laser scanning confocal microscopy of sections immunolabeled for microtubule-associated protein-2 (MAP-2) and synaptophysin. Qualitative and quantitative analysis of confocal images revealed that memantine treatment substantially decreased neuropathology in gp120 transgenic mice; this included statistically significant improvements in both dendritic and presynaptic terminal density. These results provide in vivo evidence that gp120 can activate neurotoxic pathways that can ultimately result in aberrant NMDA receptor stimulation and neuronal damage in the CNS. They also suggest that clinically tolerated NMDA receptor antagonists may be useful in the prevention of neuronal damage in HIV-1-infected patients.     

l-[H]Glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis

The anatomical distribution ofl-[³H]glutamate binding sites was determined in the presence of various glutamate analogues using quantitative autoradiography. The binding ofl-[³H]glutamate is accounted for the presence of 3 distinct binding sites when measured in the absence of Ca²⁺, Cl⁻ and Na⁺ ions. The anatomical distribution and pharmacological specificity of these binding sites correspond to that reported for the 3 excitatory amino acid binding sites selectively labeled byd-[³H]2-amino-5-phosphonopentanoate (d-[³H]AP5), [³H]kainate ([³H]KA) and [³H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([³H]AMPA) which are thought to be selective ligands for the N-methyl-d-aspartate (NMDA), KA and quisqualate (QA) receptors, respectively.     

Melatonin blocks the induction of long-term potentiation in an N-methyl-d-aspartate independent manner

Perfusion of 100 μM melatonin had no effect on low frequency synaptic transmission, but prevented the induction of tetanically induced long-term potentiation (LTP) when recorded in the dendritic region of the CA1 in rat hippocampal slices. Perfusion of 100 μM melatonin in this preparation had no effect on the multiple population spikes recorded in Mg²⁺-free medium, and, in grease-gap recordings from the CA1-subiculum slice, 100 μM melatonin had no effect on depolarisations evoked by N-methyl-d-aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). This suggests that melatonin has the ability to prevent the formation of LTP, and that this effect is not mediated by blockade of NMDA receptors.     

Epileptiform bursts elicited in CA3 hippocampal neurons by a variety of convulsants are not blocked by N-methyl-d-aspartate antagonists

Intracellular and extracellular recordings from CA₃ hippocampal neurons in vitro were used to study the ability of several NMDA (N-methyl-d-aspartate) receptor antagonists to suppress epileptiform bursts induced by NMDA and convulsants not thought to act at NMDA receptors. The antagonists, APV (d-2-amino-5-phosphonovalerate), AP-7 (d,l-2-amino-7-phosphonoheptanoate) and CPP (d,l-3-[(±)-2-car☐ypiperazin-4-yl-]-propyl-1-phosphonic acid), blocked the spontaneous and evoked bursts induced by NMDA. CPP, but not APV or AP-7, prevented the development of bursts induced by Mg-free medium. The NMDA antagonists failed to block bursting induced by kainate, 7 mM K⁺, mast cell degranulating peptide, anoxia or spontaneous bursting. In some cases the NMDA antagonists induced spontaneous bursts or enhanced burst frequency, a proconvulsant effect. It is concluded that activation of NMDA receptors is sufficient but not necessary for burst generation in the CA₃ region.     

Stimulation of α4β2 nicotinic acetylcholine receptors inhibits β-amyloid toxicity

We examined the effects of nicotinic receptor agonists against beta amyloid (Aβ) cytotoxicity to rat cortical neurons. Administration of nicotine protected against Aβ-induced neuronal death. This neuroprotection was blocked by dihydro-beta-erythroidine, an α4β2 nicotinic receptor antagonist. Furthermore, incubation with cytisine, a selective α4β2 nicotinic receptor agonist, inhibited Aβ cytotoxicity. These results suggest that α4β2 nicotinic receptor activation plays an important role in neuroprotection against Aβ cytotoxicity.     

Glutamate antagonists applied to midbody spinal cord segments reduce the excitability of the fictive rostral scratch reflex in the turtle

Glutamate antagonists applied to the cutaneous-processing region of the rostral scratch circuit in turtles reduced the excitability of the rostral scratch reflex. Segments D3-D6 (D3 = 3rd postcervical) of the midbody spinal cord receive cutaneous afferents from the rostral scratch receptive field and perform the initial integration of this cutaneous sensory input. These cutaneous-processing segments are located anterior to the rostral scratch motor pattern generator that resides mainly in segments D7-D10 located in and near the hindlimb enlargement. We prepared 1 or 2 of the midbody segments for bath application of glutamate antagonists in preparations with a complete transection of the spinal cord anterior to segment D3. Each preparation was immobilized by neuromuscular blockade and fictive scratch motor output was recorded from hindlimb muscle nerves. Application of the NMDA N-methyl-D-aspartate) antagonist APV (D-2-amino-5-phosphonovaleric acid, 50 microM) to a midbody segment significantly reduced the motor burst frequency of rostral scratch responses evoked by 3-Hz electrical stimulation of a site in that segment's dermatome. These data suggest that NMDA receptors contribute to cutaneous processing in the rostral scratch circuit. Application of APV to a midbody segment also reduced the magnitude of temporal summation in the scratch circuit in response to electrical stimuli delivered to the shell at 4- to 5-s intervals. Temporal summation was monitored at the level of hindlimb motor output as well as at the level of unit activity from 'long-afterdischarge' neurons in the midbody segments. Our observations are consistent with the hypothesis that NMDA receptors contribute to the prolonged activation of 'long-afterdischarge' neurons and the multisecond storage of excitation in the scratch reflex pathway.     

Mild mitochondrial inhibition in vivo enhances glutamate-induced neuronal damage through calpain but not caspase activation: role of ionotropic glutamate receptors

Glutamate neurotoxicity is exacerbated when energy metabolism is impaired. In vitro studies show that neuronal death in these conditions is related to mitochondrial dysfunction, ATP depletion, and the loss of calcium homeostasis. We have recently observed that, in vivo, enhancement of glutamate toxicity elicited by previous mitochondrial inhibition does not involve severe ATP depletion, suggesting the involvement of other processes. Factors such as the activation of different proteases may determine the extent and type of cell death. Protease activation might be triggered by internal or external factors, such as mitochondrial damage or the activation of a particular glutamate receptor subtype. In the present study we aimed to investigate whether moderate inhibition of mitochondrial metabolism facilitates glutamate toxicity through caspase-3 or calpain activation, as well as the contribution of NMDA and non-NMDA glutamate ionotropic receptors to this activation. Rats were pre-treated with a subtoxic dose of 3-NP and 4 h later intrastriatally injected with glutamate. Results show that neither of these treatments alone (3-NP or Glu) or in combination (3-NP+Glu) activated caspase-3. Conversely, calpain activity is induced after glutamate injection both in intact and 3-NP pre-treated rats. Inhibition of calpain activity by MDL-28170 significantly prevented striatal damage. NMDA and non-NMDA receptors contributed equally to calpain activation and to the induction of neuronal death. Results suggest that enhancement of glutamate toxicity due to inhibition of mitochondrial metabolism in vivo, does not recruit caspase-dependent apoptosis but favors calpain activation through the stimulation of both subtypes of glutamate ionotropic receptors.     

Sympathetically-mediated cardiovascular responses induced by neurochemical microinjection in the brainstem lateral tegmental field of cat

This study examines sympathetically-mediated cardiovascular responses arising from microinjections of AMPA and kynurenic acid in the medullary lateral tegmental field (LTF). The resulting behavior, which is predominately inhibitory, is nearly identical to that reported for the para-ambiguual area lying just lateral to the LTF, and suggests that the two regions may share other cardiovascular control functions.     

Cognitive impairment and increased brain neurosteroids in adult rats perinatally exposed to low millimolar blood alcohol concentrations

Epidemiological evidence suggests that adolescents and adults perinatally exposed to alcohol, even at low doses, show high prevalence of cognitive impairment and social behavior deficits, which may be in part related to alcohol-induced changes of the gamma-aminobutyric acid (GABA)ergic neurotransmission. The endogenous neurosteroid 3alpha-hydroxy,5alpha-pregnan-20-one (3alpha,5alpha-tetrahydroprogesterone/3alpha,5alpha-THP), a potent positive allosteric modulator of GABA(A) receptor function, is implicated in the physiological tuning of GABA-mediated fast inhibition and in various alcohol's actions in the brain. This study was undertaken to determine whether perinatal exposure to low millimolar blood alcohol concentrations alters cognitive skills (social discrimination and inhibitory avoidance tests), emotional reactivity (elevated plus maze test), and neurosteroid content in brain cortex and hippocampus of adult male offspring. Dams had access to a 3% alcohol solution or to an equicaloric sucrose solution from gestational day 15 to postnatal day 9. Eighty-day old alcohol-exposed male offspring exhibited impaired social recognition memory, but unchanged inhibitory avoidance performance and normal behavior on the elevated-plus maze. The concentrations of 3alpha,5alpha-THP and its precursor progesterone were more than doubled in brain cortex and hippocampus of alcohol-exposed rats, whereas in plasma only progesterone was increased. Thus, exposure to low millimolar blood alcohol concentrations has a long-lasting impact on the developing brain as it causes an impairment of social recognition as well as an increase of brain neurosteroid content in mature animals. The latter may be consequent to altered expression/activity of brain steroidogenic enzymes, as reflected by the enduring increase of the GABA(A) receptor-active neurosteroid 3alpha,5alpha-THP in brain cortex and hippocampus, but not in plasma. It is speculated that, by inducing a greater amplification of GABA(A) receptor function, the elevation of 3alpha,5alpha-THP brain content contributes to the cognitive impairment exhibited by adult alcohol-exposed offspring.     

Astroglial ablation prevents MPTP-induced nigrostriatal neuronal death

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a potent neurotoxin which destroys nigrostriatal dopamine neurons, resulting in irreversible idiopathic parkinsonism. MPTP displays dopaminergic neurotoxicity to humans, monkeys, cats and rodents. The oxidative conversion of MPTP to 1-methyl-4-phenylpyridine (MPP⁺) is responsible for the generation of its neurotoxicity. This metabolism is mediated by the action of monoamine oxidase B, which in the substantia nigra pars compacta (SNc) is localized specifically in astroglia. Employing various combinations of intra-SNc injections of MPTP and the astroglia-specific toxin, l-α-aminoadipic acid (l-α-AA), were examined the effects of selective astroglial ablation on MPTP-induced nigrostriatal neuronal death in the rat. Varying nigrostriatal cell loss was assessed primarily by the aid of fluorescent retrograde axonal tracing. Treatment with MPTP alone caused tremendous nigrostriatal cell loss, while intra-SNc co-injections of MPTP and l-α-AA produced protection against MPTP neurotoxicity in a dose-dependent fashion. Similar effects of l-α-AA occurred in the SNc pretreated with the gliotoxin just prior to or 1 day before MPTP administration. However, this preventive action by l-α-AA was considerably reduced 3 days after its intra-SNc injection. Interestingly, 7 days following l-α-AA pretreatment, nigrostriatal cell loss was even enhanced rather than attenuated by MPTP administered into the SNc. Thus, our data provide clear morphological evidence for the critical importance of the presence of astroglia in the onset of MPTP neurotoxicity.