NMDA-Antagonist MK-801-induced neuronal degeneration in Wistar rat brain detected by the Amino-Cupric-Silver method

The neurotoxic effect following a single intraperitoneal injection of MK-801 (10 mg/kg) in adult female Wistar rats at different survival times was studied with the 1994 version of de Olmos' Amino-Cupric-Silver (A-Cu-Ag) technique for detection of neural degeneration. In addition to the well documented somatodendritic degeneration observable in cortical olfactory structures, dentate gyrus, retrosplenial and sensory cortices, we detected this type of neuronal degeneration also in the main olfactory bulb, motor and anterior cingulate cortices, thalamus and cerebellum. Terminal degeneration, not reported by previous authors, was detected in cortical olfactory structures, hippocampal formation, sensory, infralimbic, prelimbic, agranular insular, ectorhinal, perirhinal and lateral orbital cortices. These results demonstrate that the A-Cu-Ag procedure is more efficient than other silver methods for detecting the degeneration induced by MK-801. In fact, the use of the A-Cu-Ag method has made it possible to infer the connectional relations between the damaged cell bodies and corresponding terminal degeneration. Our results also indicate that the A-Cu-Ag technique may be a suitable method for the staining of neurons undergoing apoptotic-like degeneration. The probable degenerative mechanism of MK-801 in the main olfactory system is discussed.     

Systemic administration of MK-801 protects against N-methyl-D-aspartate- and quisqualate-mediated neurotoxicity in perinatal rats

MK-801, a non-competitive antagonist of N-methyl-D-aspartate-type glutamate receptors, was tested for its ability to antagonize excitotoxic actions of N-methyl-D-aspartate or quisqualic acid injected into the brains of seven-day-old rats. Stereotaxic injection of N-methyl-D-aspartate (25 nmol/0.5 microliters) or quisqualic acid (100 nmol/1.0 microliter) into the corpus striatum under ether anesthesia consistently produced severe unilateral neuronal necrosis in the basal ganglia, dorsal hippocampus and overlying neocortex. The distribution of the damage corresponded to the topography of glutamate receptors in the vulnerable regions demonstrated by previous autoradiographic studies. N-Methyl-D-aspartate produced severe, confluent neuronal destruction while quisqualic acid typically caused more selective neuronal necrosis. Intraperitoneal administration of MK-801 (0.1-1.0 mg/kg) 30 min before N-methyl-D-aspartate injection had a prominent dose-dependent neuroprotective effects as assessed morphometrically by comparison of bilateral striatal, hippocampal and cerebral hemisphere cross-sectional areas five days later. A 1 mg/kg dose of MK-801 given as pre-treatment completely protected the infant brain. The same dose of MK-801 was also completely protective when administered 30 or 40 min after N-methyl-D-aspartate and afforded partial protection when given 2 h later. MK-801 pre-treatment also prevented the electrically confirmed behavioral seizures induced by N-methyl-D-aspartate. The drug significantly reduced striatal but not hippocampal or neocortical injury when given as two doses (1 mg/kg) 30 min prior to and immediately following quisqualic acid injection. The data indicate that systemic administration of MK-801 can prevent N-methyl-D-aspartate-induced neuronal injury in perinatal rat brain even when administered after the initial insult. MK-801 also partially antagonized quisqualic acid-mediated neurotoxicity, suggesting that quisqualic acid-induced toxicity is, in part, mediated through N-methyl-D-aspartate receptor activation. The sensitivity of the developing brain to the toxicity of N-methyl-D-aspartate provides a sensitive and reproducible in vivo model for exploring these issues and for screening prospective neuroprotective drugs that act at the N-methyl-D-aspartate receptor-channel complex.     

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。     

MK-801 prevents brain lesions and delayed-nonmatching-to-sample deficits produced by pyrithiamine-induced encephalopathy in rats.

Rats were trained on a spatial delayed-nonmatching-to-sample (DNMTS) task and assigned by block randomization to one of four treatments: pyrithiamine-induced thiamine deficiency (PTD), PTD with administration of MK-801 after 12 days, control with MK-801 treatment, and control without MK-801. After 15 days of treatment followed by 21 days of recovery, the PTD rats showed significant deficits for DNMTS accuracy at retention intervals (RI) that ranged from 3.0 s to 15.0 s, the RIs that produced 75% accuracy on DNMTS in staircase training, and the rate at which a novel radial arm maze task was learned. The PTD-treated rats had consistent lesions in the thalamus and the mammillary bodies. MK-801 protected rats from both behavioral deficits and brain lesions (assessed quantitatively and qualitatively) that were produced by the PTD treatment.     

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.     

MK-801对新生鼠脑外伤性神经变性的超微结构的影响

目的:研究非竞争性N-甲基-D-天门冬氨酸受体拮抗剂MK-801对新生鼠创伤性颅脑损伤(TBI)后同侧顶叶皮质和海马神经元超微结构的影响.方法:新生7 d SD大鼠,被随机分成正常对照组和实验组(实验组给予MK-801 1 mg/kg,并且细分为创伤前30 min给药、创伤即刻给药和创伤后30 min给药3个亚组).造模24 h取材,透射电镜下观察神经细胞超微结构的变化.结果:创伤后30 min给药的神经细胞出现胞质和核染色质的浓缩深染,胞质内充满大小不等的空泡;创伤即刻组的神经细胞胞质内线粒体有肿胀,但胞核的变化不明显.结论:适时和适量运用MK-180能延迟神经细胞核染色质和粒线体的变性损伤,使之停留在损伤早期,为临床联合其他药物治疗赢得时间.     

Peripheral administration of an N-methyl-D-aspartate receptor antagonist (MK-801) changes dorsal horn neuronal responses in rats

Due to the discovery of peripheral N-methyl-D-aspartate (NMDA) receptors, the effects of peripherally administrated MK-801, a non-competitive NMDA receptor antagonist, and phosphate buffered saline were tested by using the response changes of wide-dynamic range cells in the lumbar enlargement of the spinal cord in Sprague-Dawley rats. MK-801 (1 microM, 50 microl) administered directly into the subcutaneous tissue of the receptive field (n = 7), produces a reversible reduction of responses to noxious and innocuous stimuli by a peripheral action. There was no change in the responses to cutaneous stimuli following injection of phosphate buffered saline (n = 7) or following administration of MK-801 into the contralateral foot (n = 7). The present study suggests that MK-801 produces a local anesthetic like effect in the peripheral tissue.     

Dose-dependent effect of MK-801 on the levels of neuropeptides processing enzymes in rat brain regions

The appropriate levels of neuropeptides and their processing enzyme activities are required to continue a normal cell life, and the dysfunction of these peptides and enzymes are responsible for many neuronal abnormalities. Systemic administration of (+) MK-801 (dizocilpine maleate), a noncompetitive N-methyl-[D]-aspartate (NMDA) receptor antagonist, causes both neuroprotective and neurotoxic activities depending on doses and conditions. In the present study, we investigated the dose dependent effect of (+) MK-801 on prolyl endopeptidase (PEP), endopeptidase EC 24.15 (EP 24.15) and beta-D-glucuronidase activities as well as the protein levels of EP 24.15 and neuron specific enolase (NSE) in the posterior cingulate/retrosplenial cortices (PC/RSC), hippocampus, frontal cortex and striatum of female rats 3 days after the treatment. The activity of PEP was significantly increased compared with controls (saline) in the PC/RSC at 1.0 and 5.0 mg/kg doses, and in the frontal cortex at 5.0 mg/kg dose. beta-D-Glucuronidase activity was dose-dependently increased in all brain regions examined. The activity of EP 24.15 was unchanged in all regions after the treatment, whereas the Western blot analysis for EP 24.15 showed the increased protein level in the PC/RSC. These results suggest that a low dose treatment with MK-801 causes neurotoxicity in the PC/RSC and hippocampus, and the high dose treatment causes neurotoxicity in all the brain regions examined.     

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.     

Co-administration of MK-801 and morphine attenuates neuropathic pain in rat

Partial peripheral nerve injury often leads to chronic pain states, including allodynia and hyperalgesia. The purpose of this study was to investigate the involvement of the N-methyl-D-aspartate and opioid receptors in the behavioural responses following chronic constriction nerve injury (CCI). The animals were injected a combination of MK-801 (0.3 mg/kg, 20 min before, and 6 h after the operation) and morphine (8 mg/kg, 30 min prior to the operation) and were tested for allodynia and hyperalgesia reactions at 0, 3, 7, 14, 21 and 28 days after CCI. Compound action potentials were also recorded from the injured nerve 2 weeks post-operation to indicate nerve injury state electrophysiologically. Our results indicate that the CCI model importantly influences the behavioural responses to both the thermal and mechanical stimulations. Also, the pre-emptive co-administration of MK-801 and morphine has suppressive effects on the cold allodynia but a slight alleviation on the mechano-allodynia and heat hyperalgesia.     

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.     

Rescue of motoneurones by MK-801 innervating partially denervated rat muscles

Partial denervation of the extensor digitorum longus muscle by cutting the L4 spinal nerve in 3-day-old rats causes some of the remaining uninjured motoneurones to die. A previous study has shown that of the 12 motor units usually present in the remaining uninjured L5 spinal nerve, a significantly smaller number of motor units to extensor digitorum longus muscle is found in animals operated on at 3 days. This reduction can be caused by a greater sensitivity of neonatal motoneurones with axons in a partially denervated muscle, to excitotoxic effects of glutamate. Therefore an N-methyl-D-aspartic acid (NMDA) receptor antagonist, MK-801, was injected daily for 12 days after partial denervation at 3 days. Two months after the operation contractile properties, motor unit numbers and sizes were studied. Following MK-801 treatment, the reduction in muscle weight and force output of the partially denervated muscle was less than that in the untreated group. Moreover there were more motor units in MK-801 treated animals. After partial denervation only, 15% of the total number of motor units was present whereas when the same operation was followed by treatment with MK-801, 29% remained. The mean motor unit size in the untreated group was 69% while after treatment with MK-801 it was 152% of the control. Thus treatment with MK-801 after partial denervation of neonatal animals rescued some of the motoneurones destined to die, and allowed expansion of motor unit territory of most of the surviving motoneurones. Electronic Publication     

Protective effects of MK-801 on manganese-induced glutamate metabolism disorder in rat striatum

Manganese (Mn) is one of the ubiquitous environmental pollutants that can induce an indirect excitotoxicity caused by altered glutamate (Glu) metabolism. The present study has been carried out to investigate the mechanisms of Glu metabolism disorder and the neuroprotective role of MK-801, a non-competitive N-methyl-d-aspartate receptor antagonist, against Mn-induced excitotoxicity in rat striatum. Fifty rats were randomly divided into five groups with 10 animals in each group: control group, Mn-treated group (8, 40, and 200 μmol/kg), and MK-801-pre-treated group. Administration of MnCl₂·6H₂O at dose of 200 μmol/kg body weight for 4 weeks significantly increased the concentrations of Glu and Mn in the striatum (P<0.01). In addition, Mn also increased the activity of phosphate-activated glutaminase (PAG) (54.38%, P<0.01), enhanced striatum cell apoptosis rate (65.04%, P<0.01) and decreased the activity of glutamine synthetase (GS) (28.88%, P<0.01). Pre-treatment with MK-801 at a dose of 0.3 μmol/kg body weight for 4 weeks prior to 200 μmol/kg Mn administration prevents the alterations of the activities of PAG and GS and concentrations of glutamine (Gln). In addition, pre-treatment with MK-801 significantly reduced the striatum cell apoptosis rate. In conclusion, the results suggest that MK-801 possesses the ability to attenuate Mn-induced Glu metabolism disorder in the striatum through mechanisms involving disruption enzyme activities of GS and PAG.     

MK-801, a non-competitive antagonist of NMDA receptor, prevents methamphetamine-induced decrease of striatal dopamine uptake sites in the rat striatum.

We investigated the effects of MK-801, a non-competitive antagonist of NMDA receptor, on methamphetamine-induced decrease in dopamine (DA) uptake sites in the rat striatum. Repeated administrations of an escalating dose of methamphetamine (2.5, 5, 7.5, 10 mg/kg s.c. x2, every other day for a week) produced decreased DA uptake sites assayed by binding with [3H]GBR 12935 in the striatum. Co-administration of MK-801 and methamphetamine significantly prevented the methamphetamine-induced decrease in striatal [3H]GBR 12935 binding. Administration of MK-801 alone did not affect [3H]GBR 12935 binding. These results suggest that some neurochemical effects of methamphetamine may be mediated via mechanism involving excitatory amino acids.     

Regional distribution and properties of [3H]MK-801 binding sites determined by quantitative autoradiography in rat brain

[3H]MK-801 binding in rat brain was characterized using a quantitative autoradiographic binding assay. [3H]MK-801 binding (5 nM) reached equilibrium by 120 min at 23 degrees C. [3H]MK-801 appeared to label a single high affinity site with an affinity constant of approximately 11 nM. [3H]MK-801 binding was heterogeneously distributed throughout the brain with the following order of binding densities: hippocampal formation greater than cortical areas greater than striatum greater than thalamus. Competitive N-methyl-D-aspartate antagonists, DL-2-amino-5-phosphonopentanoic acid, DL-2-amino-7-phosphonoheptanoic acid, 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, and cis-4-phosphonomethyl-2-piperidine carboxylic acid, inhibited [3H]MK-801 binding. Glycine antagonists, 7-chlorokynurenic acid and kynurenic acid, also inhibited [3H]MK-801 binding. Furthermore, the inhibition of [3H]MK-801 binding by the quinoxalinedione compounds 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione was reversed by glycine. [3H]MK-801 binding was also inhibited by zinc ions [3H]MK-801 binding was enhanced by glycine or N-methyl-D-aspartate. These results demonstrate that [3H]MK-801 can be used in a quantitative autoradiographic assay as a functional probe for the N-methyl-D-aspartate receptor complex.     

Sex differences and influence of gonadal hormones on MK801-induced neuronal degeneration in the granular retrosplenial cortex of the rat

MK801, PCP, and ketamine are non-competitive NMDA receptor-antagonists drugs that in humans produce psychomimetic effects and neurocognitive disturbances reminiscent to those of schizophrenia. The administration of these drugs in animals has been used as a pharmacological model to study the NMDA receptor hypofunction-hypothesis of schizophrenia. In animals, the biological effect of MK801 is dose-dependent. Low doses induce behavioral disturbances and higher doses, in addition, promote neurotoxicity in many brain regions, particularly the granular retrosplenial cortex (RSG). The neurotoxic effect of MK801 is sexually dimorphic, being females markedly more sensitive than males; however, the involvement of gonadal hormones is elusive. Here we show that a single intraperitoneal injection of 5 mg/kg of MK801 induced overt neurodegeneration in RSG of female rats, including abundant somatic degeneration in layer 4, and somatodendritic and terminal degeneration in layers 1, 4, and 5. MK801-degeneration in males was scarce and mainly evidenced by the presence of few argirophilic somas in layer 4. Ovariectomized rats were not significantly different than intact females, while orchiectomized rats showed robust MK801-toxicity. Testosterone and dihydrotestosterone (DHT) inhibit MK801-toxicity in orchiectomized rats. In ovariectomized rats only DHT, but not testosterone, prevented MK801-induced degeneration, while in intact females, DHT was only partially protective. Treatment of intact males with estradiol benzoate significantly enhanced MK801-toxicity. Altogether, our experiments indicate that non-aromatizable androgens protect RSG from MK801-toxicity, while estrogens counteract this protection. Thus, the balance of androgens and estrogens delineate the sexual dimorphism of the RSG to the toxic effect of MK801.     

Stereoselective antagonism of NMDA-stimulated noradrenaline release from rat hippocampal slices by MK-801

N-Methyl-D-aspartate (NMDA)-stimulated [3H]noradrenaline release from rat hippocampal slices was blocked stereospecifically and non-competitively by MK-801 with the (+)-isomer achieving 50% blockade of 100 microM NMDA at 16 nM. The results indicate that MK-801 is the most potent NMDA antagonist yet described and that it blocks NMDA-stimulated neurotransmitter release by an action at the so-called 'phencyclidine (PCP) site'.     

N-methyl-D-aspartate-induced excitotoxicity in adult rat retina is antagonized by single systemic injection of MK-801

Intravitreal injection of N-methyl-D-aspartate (NMDA) produced a substantial damage to the adult rat retina that was largely restricted to inner retinal layers, including the ganglion cell layer (GCL), inner nuclear layer (INL), inner, and outer plexiform layers. This retinal damage was significantly reduced by a systemic injection of a low dose of MK-801 (0.5 mg/kg), a potent NMDA-receptor antagonist. This neuroprotection was dose dependent and was most effective when the antagonist was given 1 h before NMDA insult. An intraperitoneal injection of 0.5 mg/kg MK-801 provided a virtually complete protection to the retina to the NMDA-induced toxicity, as indicated quantitatively by the number of DiI-filled retinal ganglion cells, the number of cells in the GCL and INL that undergo DNA fragmentation, and the edematous changes in retinal thickness. A post-lesion administration of MK-801 was still able to provide an effective neuroprotective effect to the retina, but this protection was lost when MK-801 was given 4 h after NMDA exposure. The current results indicate a therapeutic potential of systemic application of MK-801 in protecting the adult rat retina from neurologic disorders related to excessive activation of NMDA receptors.     

MK-801 blocks NMDA receptor-mediated synaptic transmission and long term potentiation in rat hippocampal slices

The effect of the anticonvulsant and neuroprotective agent (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine maleate (MK-801) has been studied on synaptic events in the CA1 region of rat hippocampal slices. MK-801 blocked selectively the N-methyl-D-aspartate receptor-mediated component of synaptic transmission, which can be recorded in response to single shock stimulation of the Schaffer collateral-commissural pathway in the absence of added Mg2+ to the perfusate. MK-801 also prevented the induction of long term potentiation, which is normally produced in this pathway by high frequency stimulation in the presence of Mg2+.     

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.