MK-801 induces c-fos protein in thalamic and neocortical neurons of rat brain

MK-801, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, leads to a dramatic induction of c-fos-like protein in neurons in deep layers of the neocortex, in dorsal and ventral midline thalamic nuclei and in neurons in the central grey of rat brain. This induction of c-fos by MK-801 is dose-and time-dependent occurring within 2 h and dissipating by 24 h after injection (0.5-8.0 mg/kg, i.p.). The mechanism of this paradoxical induction of c-fos by MK-801 is unclear; however, the pattern of induction appears to follow the distribution of the antagonist-preferring NMDA receptor site.     

Protein Synthesis Is Required for Induction of Amnesia Elicited by Disruption of the Reconsolidation of Long-Term Memory

Studies on common snails previously trained to an associative skill consisting of rejecting a defined foodstuff addressed the effects of NMDA glutamate receptor antagonists (MK-801 and APV) and protein synthesis inhibitors (cycloheximide and anisomycin) on long-term memory reconsolidation processes. Injections of each of the study compounds before the reminding procedure 24 h after training were found to lead to impairment of the reproduction of the acquired skill, which lasted at least three weeks. Repeat training of these animals to reject the same foodstuff as used in the initial training did not lead to acquisition of the skill. However, simultaneous injections of a protein synthesis inhibitor and an NMDA receptor antagonist (MK-801 + cycloheximide or APV + anisomycin) did not impair the skill. In subsequent experiments, snails received cycloheximide at different times after exposure to MK-801/reminding. Administration of cycloheximide 3 and 6 h after MK-801/reminding led to the development of incomplete amnesia and repeat training of the animals led to rapid restoration of memory. Administration of cycloheximide 9 h after MK-801/reminding evoked the development of stable amnesia characterized by impairment of skill formation on repeat training. We propose that the mechanisms of amnesia induced by the NMDA glutamate receptor antagonist, by analogy with the mechanisms of other long-term adaptive rearrangements of the brain, depend on translation and can be suppressed by inhibitors of translation. The “time window” for the dependence of amnesia induction processes on the synthesis of protein molecules was 6–9 h after exposure to MK-801/reminding.     

3H]MK-801 binding in immature and mature chicken forebrain.

Previous studies have shown that the rate of calcium uptake stimulated by N-methyl-D-aspartate (NMDA) increased during the maturation phase of synapse development in chicken forebrain. To investigate whether this change in function is due to a change in the properties of NMDA receptor associated ion channels, we measured the binding of [3H]MK-801 (a ligand which binds to the NMDA receptor associated ion channel) to membranes from immature and mature chicken brain. The binding properties of MK-801 in chicken brain were similar to those in mammalian brain. There was no significant difference in any of the binding parameters measured at the two ages, i.e. KD, Bmax and optimal glutamate concentration for and maximal enhancement by glutamate of MK-801 binding. These results suggest that there is no change in the NMDA operated ion-channels during maturation. Thus the maturational change in NMDA receptor function could be due to: a change in the agonist portion of the NMDA receptor, a change in the regulation of the receptor/ionophore complex, perhaps by the postsynaptic density whose structure and composition changes during the same period, or a change in the number of voltage-sensitive calcium channels recruited as a result of NMDA receptor activation.     

MK-801对新生大鼠脑外伤后神经元凋亡的影响

目的：探讨N-甲基-D-天冬氨酸(NMDA)受体拮抗剂MK-801对新生大鼠创伤性脑外伤(traumatic brain injury,TBI)后神经元凋亡的影响。方法：建立新生7 d大鼠顶叶皮质挫伤模型,在TBI前30 min、TBI后即刻、TBI后30 min分别给予腹腔注射MK-8011 mg/kg,在TBI后24 h取脑,连续切片,行H-E染色和Caspase-3免疫组化染色,检测脑神经元细胞的损伤和凋亡。结果：MK-801三组不同时间用药组与TBI组相比,在创伤同侧的扣带皮质、顶叶皮质和丘脑神经元凋亡细胞数减少,有显著性差异。其中TBI后即刻用MK-801治疗效果最好。结论：MK-80l能明显减少TBI后神经元的凋亡。     

The amplitude of N2pc reflects the physical disparity between target item and distracters

The non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist (+)MK-801 is widely used in animal research (over 3000 publications), however its extracellular brain concentration has never been reported. Here, we show using in vivo microdialysis that systemic injection of (+)MK-801 at doses of 0.05, 0.1 or 0.2 mg/kg resulted in peak brain ECF concentration of 6, 14 or 34 nM, respectively. Moreover, (+)MK-801 resulted in a dose-dependent learning impairment in the Morris water maze as well as hyperactivity in the open field. These data demonstrate for the first time that (+)MK-801 at doses producing behavioural alterations expected from NMDA receptor blockade reaches extracellular brain concentrations corresponding to the affinity at NMDA receptors.     

Regional alterations in brain amino acids after administration of the N-methyl-D-aspartate receptor antagonists MK-801 and CGP 39551 in rats.

The effects of the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 and the novel competitive NMDA receptor antagonist CGP 39551 on levels of 11 amino acids, including several excitatory and inhibitory neurotransmitters, were studied in 12 brain regions of rats. Both drugs were administered at doses which produced comparable behavioural effects (ataxia, hyperactivity). Amino acids were determined in brain tissue by high-performance liquid chromatography after o-phthaldialdehyde precolumn derivatization. MK-801 (0.1 mg/kg, i.p.) moderately increased the concentration of glutamate and GABA in several brain regions. Other amino acids (glutamine, taurine, asparagine, alanine, serine) were only altered in single brain regions, or were not altered at all (aspartate, glycine, threonine, arginine). In contrast to MK-801, CGP 39551 (10 mg/kg, i.p.) increased glutamate levels only in the cerebellum, and produced no significant alterations in levels of GABA. The data demonstrate differences in alterations of amino acid levels in response to competitive and non-competitive NMDA receptor antagonists and support the assumption that competitive NMDA antagonists may be more selective than non-competitive antagonists.     

Partial protective effect of MK-801 on MPTP-induced reduction of striatal dopamine in mice.

The protective effect of MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced reduction of striatal dopamine (DA) was examined in C57 BL/6 mice. Striatal DA levels were significantly higher in animals receiving parenteral MK-801 before and for 48 h following MPTP administration after 28 days than in animals receiving MPTP alone. The effect was not due to inhibition of monoamine oxidase-B (MAO-B) by MK-801. These data suggest that NMDA receptors may be involved in some of the neurotoxicity produced by MPTP.     

离子型谷氨酸受体拮抗剂MK-801浓度与全脑缺血再灌注大鼠海马内源性神经干细胞的增殖**

背景：脑缺血再灌注后,过度释放的兴奋性氨基酸可通过N-甲基-D-天冬氨酸(NMDA)受体激活内源性神经干细胞,促使其增殖、分化,修复神经细胞,但同时也导致细胞内钙离子超载,引起神经细胞的损伤。 目的：观察NMDA受体拮抗剂MK-801浓度对脑缺血再灌注大鼠海马内源性神经干细胞增殖的影响。 方法：SD大鼠随机分为正常对照组、手术对照组及MK-801 0.2,0.4,0.6,0.8,1.0,1.2 mg/kg组。除正常对照组外,大鼠首先进行侧脑室插管,3 d后进行4条血管阻断方法制备大鼠全脑缺血再灌注模型。在模型制作前30 min按照不同浓度侧脑室注射MK-801。正常对照组和手术对照组侧脑室注射同剂量的生理盐水。免疫组织化学、RT-PCR技术检测各组脑海马nestin阳性细胞及其mRNA表达。 结果与结论：MK-801浓度在0.8 mg/kg以下时,用药组大鼠脑海马nestin mRNA及蛋白的表达与手术对照组差异无显著性意义(P > 0.05),呈现高表达;当MK-801浓度达到0.8 mg/kg时,与手术对照组相比,用药组大鼠脑海马nestin基因及蛋白的表达明显下降(P < 0.05),并随浓度的增高呈递减趋势。提示MK-801在浓度为0.6 mg/kg时,即可抑制钙超载保护神经元,又有良好的刺激神经干细胞增殖作用。 关键词：离子型谷氨酸受体拮抗剂;脑缺血;再灌注;神经干细胞;MK-801 doi:10.3969/j.issn.1673-8225.2012.06.012     

Effect of NMDA on Production of Reactive Oxygen Species by Human Lymphocytes

Low concentrations (<20 M) of N-methyl-D-aspartate (NMDA), an agonist of specific receptors of brain glutamatergic systems, promote the formation of reactive oxygen species (ROS) both in the whole blood and in lymphocyte fraction. Further increase in NMDA concentrations led to progressive increase in ROS content in the whole blood, but to its decrease in lymphocyte suspension. The activating effect of NMDA is abolished by antioxidant N-acetylcysteine (5 mM) and NMDA-type glutamate receptor antagonist MK-801 (5 M). Phorbol myristate acetate (PMA, 1 M) also increased ROS content in the examined structures. This effect was antagonized by N-acetylcysteine, but not MK-801.     

Role of NR2B-containing N-methyl-D-aspartate receptors in haloperidol-induced c-Fos expression in the striatum and nucleus accumbens

Administration of haloperidol in rats leads to a robust induction of immediate-early genes including c-Fos throughout the striatum, which is significantly attenuated by pretreatment with the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801. The striatum expresses mainly NR1/NR2A and NR1/NR2B subtypes of NMDA receptors, each having different functional and pharmacological properties. In this study, rats were pretreated with Ro 25-6981, a selective antagonist for NR2B-containing NMDA receptors, in order to determine the relative contribution of this NMDA receptor subtype in NMDA-dependent haloperidol-induced c-Fos expression. Furthermore, to determine whether NMDA receptor subtype dependence of haloperidol-induced c-Fos expression is unique to the binding profile of haloperidol or whether it is a property of D2 receptor antagonism, the selective D2/D3 dopamine receptor antagonist, raclopride, was also used. Pretreatment with Ro 25-6981 led to a significant reduction in the number of nuclei showing c-Fos immunoreactivity in both the medial and lateral parts of the striatum. In the medial part of the striatum, this attenuation was almost as marked as that seen following pretreatment with MK-801; however, in the lateral part MK-801 pretreatment led to a significantly greater reduction in the number of c-Fos positive nuclei than did Ro 25-6981 pretreatment. This suggests that NR2B-containing NMDA receptors are involved in mediating most of the NMDA-dependent c-Fos expression in the medial striatum, but only responsible for mediating part of this induction in the lateral striatum. Furthermore, the pattern of attenuation of raclopride-induced c-Fos expression following Ro 25-6981 pretreatment was similar to that of haloperidol-induced c-Fos expression, indicating that the NMDA receptor subtype dependence of haloperidol-induced c-Fos expression is a property of D2 antagonism. The results indicate that NR2B-containing NMDA receptors are mainly involved in mediating haloperidol-induced c-Fos expression in the medial or "limbic" striatum, and suggest that NR2A-containing NMDA receptors may preferentially mediate haloperidol induced c-Fos expression in the lateral or "motor" striatum. This may have implications in the treatment of schizophrenia because co-administration of a selective blocker of NR2A-containing NMDA receptors may be able to reduce the severity of extrapyramidal motor symptoms caused by haloperidol treatment without interfering with its therapeutic effect that is presumably mediated via the medial part of the striatum.     

Phrenic long-term facilitation requires NMDA receptors in the phrenic motonucleus in rats

Exposure to episodic hypoxia induces a persistent augmentation of respiratory activity, known as long-term facilitation (LTF). LTF of phrenic nerve activity has been reported to require serotonin receptor activation and protein syntheses. However, the underlying cellular mechanism still remains poorly understood. NMDA receptors play key roles in synaptic plasticity (e.g. some forms of hippocampal long-term potentiation). The present study was designed to examine the role of NMDA receptors in phrenic LTF and test if the relevant receptors are located in the phrenic motonucleus. Integrated phrenic nerve activity was measured in anaesthetized, vagotomized, neuromuscularly blocked and artificially ventilated rats before, during and after three episodes of 5 min isocapnic hypoxia ( P _a,O2= 30–45 mmHg), separated by 5 min hyperoxia (50% O₂). Either saline (as control) or the NMDA receptor antagonist MK-801 (0.2 mg kg⁻¹, i.p. ) was systemically injected ∼1 h before hypoxia. Phrenic LTF was eliminated by the MK-801 injection (vehicle, 32.8 ± 3.7% above baseline in phrenic amplitude at 60 min post-hypoxia; MK-801, −0.5 ± 4.1%, means ± s.e.m. ), with little change in both the CO₂-apnoeic threshold and the hypoxic phrenic response (HPR). Vehicle (saline, 5 × 100 nl) or MK-801 (10 μ m ; 5 × 100 nl) was also microinjected into the phrenic motonucleus region in other groups. Phrenic LTF was eliminated by the MK-801 microinjection (vehicle, 34.2 ± 3.4%; MK-801, −2.5 ± 2.8%), with minimal change in HPR. Collectively, these results suggest that the activation of NMDA receptors in the phrenic motonucleus is required for the episodic hypoxia-induced phrenic LTF.     

Frequency characteristics of evoked and oscillatory electroencephalic activity in a human memory scanning task

Huperzine A, a novel cholinesterase inhibitor, was found to inhibit the N-methyl-D-aspartate (NMDA) receptors in the brain. In this study, the mechanisms of the NMDA receptor inhibition were investigated using [3H]dizocilpine (MK-801) binding in synaptic membrane of rat cerebral cortex. Changing the concentrations of L-glutamate and L-glycine did not alter the potency of huperzine A. Spermidine caused rightward shift of the concentration-response curve of huperzine A, and considerably increased its IC(50) value. Huperzine A did not affect the potency of unlabeled (+)-MK-801 in [3H]MK-801 binding. Saturation binding studies reveal that huperzine A exerts a negative allosteric modulation on the MK-801 binding site within the NMDA receptor-channel. The results suggest that huperzine A is a non-competitive antagonist of the NMDA receptors, acting at one of the polyamine binding sites.     

Basal forebrain cholinergic lesions reduce heat shock protein 72 response but not pathology induced by the NMDA antagonist MK-801 in the rat cingulate cortex

Non-competitive N-methyl-D-aspartate (NMDA) antagonists, in addition to their neuroprotective potential, possess neurotoxic properties and induce seizures and psychosis. MK-801 induces cytoplasmic vacuoles and heat shock protein in pyramidal neurones in the rodent posterior cingulate and retrosplenial cortex. The mechanism of this neurotoxicity is unclear, involving many neurotransmitter systems. The aim of this study was to investigate the role of cholinergic pathways from the nucleus basalis of Meynert in mediating MK-801-induced neurotoxicity. Cholinergic projections from the nucleus basalis of Meynert were lesioned by focal injection of 192-IgG-saporin (80 ng), which after 7 days reduced the number of cholinergic cell bodies by 70% in the lesioned nucleus compared to the uninjected nucleus. Following a unilateral cholinergic lesion, MK-801 (5 mg/kg s.c.) induced expression of hsp72 mRNA (6 h) and HSP72 protein immunoreactivity (24 h) was reduced by 42 and 60%, respectively in the ipsilateral compared to the contralateral posterior cingulate. Despite this apparent protective effect, the unilateral cholinergic lesion did not affect the degree of neuronal vacuolation (6 h), necrosis (24 h) or the large and prolonged increase in cerebral blood flow which occurred over the first 9h following MK-801 administration. These results demonstrate that cholinergic neurones in the nucleus basalis of Meynert play an important role in the heat shock response to NMDA antagonist-induced neurotoxicity but also reveal an unexpected divergence between the heat shock response and the pathophysiological response. This suggests that other cholinergic pathways or non-cholinergic mechanisms are responsible for the pathological changes induced by MK-801.     

MK-801, an antagonist of NMDA receptors, inhibits injury-induced c-fos protein accumulation in rat brain

Unilateral lesions of the rat hippocampus produced by needle insertion lead to ipsilateral accumulation of c-fos protein in dentate granule cells and neurons in the piriform cortex, as well as in glial-like cells in the corpus callosum and in ependymal cells lining the lateral ventricle adjacent to the lesion site. C-fos protein was detected immunocytochemically using two different antibodies in formalin-fixed brain sections. The N-methyl-D-aspartate (NMDA) antagonist MK-801 produced a dose- and time-dependent inhibition of c-fos protein accumulation in dentate granule cells and in neurons in the piriform cortex, but did not affect glial or ependymal c-fos protein accumulation. MK-801 at 4 mg/kg injected two hours before lesion inhibited c-fos accumulation. Thus, c-fos protein accumulation in hippocampal neurons and in neurons in the piriform cortex induced after traumatic brain injury involves activation of NMDA receptors.     

The NMDA receptor participates in respiration-related mitral cell synchrony

The N-methyl-d-aspartate (NMDA)-type glutamate receptor participates in the excitation of olfactory bulb mitral cells and is important in granule-cell-mediated feedback-inhibition. In the present study, extracellular unit recordings were made in vivo to demonstrate that the firing rates of mitral cells are not affected by peripheral administration of the non-competitive NMDA receptor antagonist MK-801. However, while over 50% of odor-driven mitral cell activity is normally correlated with the respiratory cycle, only about 10% of mitral cell activity is correlated with the respiratory cycle 30 min after MK-801 administration. Thus, the NMDA receptor is a participant in normal respiration-related mitral cell activity and may have an important role in the formation of bulb oscillations that encode olfactory information. Furthermore, the NMDA receptor is in a position to mediate activity-dependent changes in the bulb that rely on synchronous activity. Received: 30 March 1997 / Accepted: 15 July 1997     

The topography of MK-801-induced locomotor patterns in rats

Locomotor patterns in rats given systemic injections of the novel, noncompetitive N-methyl-D-aspartate (NMDA) antagonist, MK-801 were characterized using Digiscan Animal Activity Monitoring System. At low systemic doses, MK-801 produced an activity pattern most similar to patterns previously described for less potent noncompetitive NMDA antagonists; this was typified by hyperactive locomotor behavior, with increases in distance travelled, speed, and clockwise/anticlockwise locomotion, and a marked decrease in rearing behavior. Although MK-801 elicited some motor patterns similar to those previously described for sympathomimetic agents, including hyperactivity and increased stereotypy, it did not produce increased rearing behavior, the most prominent sympathomimetic effect. These results demonstrated that the topography of locomotion elicited by low systemic doses of MK-801 is most similar to locomotor patterns previously described for noncompetitive NMDA receptor antagonists.     

Effect of NMDA receptor antagonist on proliferation of neurospheres from embryonic brain

The N-methyl-D-aspartate (NMDA) receptor, a subtype of ionotropic glutamate receptors, plays an important role in the regulation of neuronal development, learning and memory, and neurodegenerative diseases. NMDA receptor blockade enhances neurogenesis in the hippocampal dentate gyrus in vivo. The effect of NMDA receptor antagonist on proliferation of neural progenitor cells, however, remains to be determined. We investigated changes in the diameter and number of neurospheres derived from the embryonic rat brain after NMDA receptor blockade. Cortical progenitor cells were isolated from gestational day 18 fetal rats according to the Percoll density gradient method. Cultured spheres expressed neural progenitor markers, musashi-1 and nestin. Immunohistochemical analysis demonstrated that cells in Dulbecco's modified Eagle medium/F12 containing 1% fetal bovine serum on day 8 differentiated to MAP-2-positive neurons and GFAP-positive astrocytes. The expression of NR1 and NR2B subunits of the NMDA receptor in neurospheres was detected. Neither brief nor sustained exposure to NMDA altered the diameter and number of neurospheres. Brief exposure to 30 μM MK-801, an NMDA receptor antagonist, decreased the diameter of neurospheres. Sustained exposure to 30 μM MK-801 decreased the diameter and number of neurospheres. Our results provide evidence that MK-801 directly decreased proliferation of neural progenitor cells.     

MK-801-induced sprouting by CGRP immunoreactive primary afferent fibers in the dorsal spinal cord of the rat

In the present study, rats received daily injections of the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, over 30 consecutive days. The effects of MK-801 on the distribution of calcitonin gene-related peptide (CGRP)-immunoreactive fibers in the dorsal spinal cord of the rat were subsequently examined. In addition to the normal immunostaining pattern in laminae I, II and lateral V, a dense network of CGRP-immunoreactive fibers was observed along the medial border of the dorsal horn and within the dorsal grey commissure. This marked increase in immunoreactivity was virtually eliminated following dorsal rhizotomy. These observations suggest that MK-801 induces intraspinal sprouting by CGRP immunoreactive primary afferent fibers in vivo.     

Suppressive effect of clozapine but not haloperidol on the increases of neuropeptide-degrading enzymes and glial cells in MK-801-treated rat brain regions

MK-801, a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, produces neurotoxicity in adult rodent brain, and causes schizophrenia-like psychosis and cognitive dysfunction. Since neuropeptides and neuropeptide-degrading enzymes play important roles in cognitive function, we examined whether or not MK-801-induced schizophrenia-like psychosis is co-related with the changes of these enzymes in rat brain regions. In the present study, we investigated the effect of systemic treatment with MK-801 (0.5mg/kg) on neuropeptide-degrading enzymes, prolyl oligopeptidase (POP) and thimet oligopeptidase (EP 24.15), and glial marker proteins GFAP and CD11b in rat brain regions. The levels of POP and EP 24.15 activities increased significantly three days after treatment with MK-801 in the posterior cingulate/retrosplenial cortices (PC/RSC). Since atypical neuroleptic clozapine but not typical neuroleptic haloperidol prevents the MK-801-induced schizophrenia-like symptoms, we further examined the pretreated effects of the neuroleptics. Clozapine, but not haloperidol, significantly attenuated MK-801-induced changes in the levels of the neuropeptide-degrading enzymes. Immunohistochemical studies on GFAP and CD11b showed the increase in the PC/RSC of MK-801-treated rat brain and the pretreatment with clozapine suppressed these changes. Double immunostain experiments of EP 24.15 and GFAP antibodies demonstrated some co-localization of the neuropeptidase with astrocytes. The present findings suggest that change of neuropeptidases in the brain is in part correlated with changes of glial cells, and may play an important role in the control of schizophrenia-like psychotic disorders.