DNA microarray unravels rapid changes in transcriptome of MK-801 treated rat brain

AIM:To investigate the impact of MK-801 on gene expression patterns genome wide in rat brain regions. METHODS:Rats were treated with an intraperitoneal injection of MK-801 [0.08(low-dose) and 0.16(highdose) mg/kg] or NaC l(vehicle control). In a first series of experiment,the frontoparietal electrocorticogram was recorded 15 min before and 60 min after injection. In a second series of experiments,the whole brain of each animal was rapidly removed at 40 min post-injection,and different regions were separated:amygdala,cerebral cortex,hippocampus,hypothalamus,midbrain and ventral striatum on ice followed by DNA microarray(4 × 44 K whole rat genome chip) analysis.RESULTS:Spectral analysis revealed that a single systemic injection of MK-801 significantly and selectively augmented the power of baseline gamma frequency(30-80 Hz) oscillations in the frontoparietal electroencephalogram. DNA microarray analysis showed the largest number(up- and down- regulations) of gene expressions in the cerebral cortex(378),midbrain(376),hippocampus(375),ventral striatum(353),amygdala(301),and hypothalamus(201) under low-dose(0.08 mg/kg) of MK-801. Under high-dose(0.16 mg/kg),ventral striatum(811) showed the largest number of gene expression changes. Gene expression changes were functionally categorized to reveal expression of genes and function varies with each brain region.CONCLUSION:Acute MK-801 treatment increases synchrony of baseline gamma oscillations,and causes very early changes in gene expressions in six individual rat brain regions,a first report.     

Comparative proteome analysis of thalamus in MK-801-treated rats

Two-dimensional gel-electrophoresis in combination with mass spectrometry is a powerful approach to compare protein expression in brain tissues. Using this proteomic approach, and based on the hypothesis that schizophrenia involves hypoglutamergic brain function, alterations in protein levels in the thalamus of rats treated with the N-methyl-D-aspartate (NMDA) receptor antagonist [+]-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cycloheptene-5,10-iminehydrogenmaleate (MK-801), as compared to saline-treated animals, were assessed in an unbiased fashion. The rats were divided into two groups; group 1 (short-term treated) and group 2 (long-term treated). In group 1, the levels of seven proteins were increased and four proteins reduced. In group 2, the levels of six proteins were reduced. Several of the altered proteins (heat shock proteins 60 and 72, albumin, dihydropyrimidinase related protein-2, aldolase c, and malate dehydrogenase) have previously been connected to schizophrenia. Alterations of other proteins (dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex E2, guanine deaminase, alpha-enolase, aconitase, ATP-synthase and alpha-internexin), have not, to the best of our knowledge, earlier been implicated in schizophrenia pathology. Our results show the high potential of using proteomic methods for the validation of animal models of schizophrenia and to identify new proteins involved in the pathophysiological mechanisms of schizophrenia.     

Pentylenetetrazol-induced seizures affect the levels of prolyl oligopeptidase, thimet oligopeptidase and glial proteins in rat brain regions, and attenuation by MK-801 pretreatment

The regulatory mechanisms of neuropeptide-metabolizing enzymes often play a critical role in the pathogenesis of neuronal damage. A systemic administration of pentylenetetrazol (PTZ), an antagonist of GABA(A) receptor ion channel binding site, causes generalized epilepsy in an animal model. In the present study, we examined the involvement of prolyl oligopeptidase (POP), thimet oligopeptidase/neurolysin (EP 24.15/16) and glial proteins in PTZ-treated rat brain regions, and the suppressive effect of MK-801, a non-competitive NMDA receptor antagonist, pretreatment for their proteins. The activity of POP significantly decreased in the hippocampus at 30min and 3h, and in the frontal cortex at 3h after PTZ treatment, and pretreatment with MK-801 recovered the activity in the cortex at 3h. The activity of EP 24.15/16 significantly decreased in the hippocampus at 3h and 1 day, and in the cortex at 3h after the PTZ administration, whereas pretreatment with MK-801 recovered the change of the activity. The Western blot analysis of EP 24.15 showed significant decrease of the protein level in the hippocampus 3h after the PTZ treatment, whereas pretreatment with MK-801 recovered. The expression of GFAP and CD11b immunohistochemically increased in the hippocampus of the PTZ-treated rat as compared with controls. Pretreatment with MK-801 also recovered the GFAP and CD11b expression. These data suggest that PTZ-induced seizures of the rats cause indirect activation of glutamate NMDA receptors, then decrease POP and EP 24.15/16 enzyme activities and EP 24.15 immunoreactivity in the neuronal cells of the hippocampal formation. We speculate that changes of those peptidases in the brain may be related to the levels of the neuropeptides regulating PTZ-induced seizures.     

Modulation of the levels of NMDA receptor subunit mRNA and the bindings of [3H]MK-801 in rat brain by chronic infusion of subtoxic dose of MK-801

The effects of continuous infusion of NMDA receptor antagonist MK-801 on the modulation of NMDA receptor subunits NR1, NR2A, NR2B, and NR2C were investigated by using in situ hybridization study. Differential assembly of NMDA receptor subunits determines their functional characteristics. Continuous intracerebroventricular (i.c.v.) infusion with MK-801 (1 pmol/10 l/h) for 7 days resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels without producing stereotypic motor syndromes. The levels of NR1 mRNA were significantly increased (9-20%) in the cerebral cortex, striatum, septum, and CA1 of hippocampus in MK-801-infused rats. The levels of NR2A mRNA were significantly decreased (11-16%) in the CA3 and dentate gyrus of hippocampus in MK-801-infused rats. In contrast to NR2A, NR2B subunit mRNA levels were increased (10-14%) in the cerebral cortex, caudate putamen, and thalamus. However, no changes of NR2C subunits in cerebellar granule layer were observed. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased (12-25%) significantly in almost all brain regions except in the thalamus and cerebellum after 7 days infusion with MK-801. These results suggest that region-specific changes of NMDA receptor subunit mRNA and [³H]MK-801 binding are involved in the MK-801-infused adult rats.     

NMDA glutamate receptor antagonist MK-801 induces proteome changes in adult human brain slices which are partially counteracted by haloperidol and clozapine

Deciphering the molecular pathways associated with N-methyl-D-aspartate receptor (NMDAr) hypofunction and its interaction with antipsychotics is necessary to advance our understanding of the basis of schizophrenia, as well as our capacity to treat this disease. In this regard, the development of human brain-derived models that are amenable to studying the neurobiology of schizophrenia may contribute to filling the gaps left by the widely employed animal models. Here, we assessed the proteomic changes induced by the NMDA glutamate receptor antagonist MK-801 on human brain slice cultures obtained from adult donors submitted to respective neurosurgery. Initially, we demonstrated that MK-801 diminishes NMDA glutamate receptor signaling in human brain slices in culture. Next, using mass-spectrometry-based proteomics and systems biology in silico analyses, we found that MK-801 led to alterations in proteins related to several pathways previously associated with schizophrenia pathophysiology, including ephrin, opioid, melatonin, sirtuin signaling, interleukin 8, endocannabinoid, and synaptic vesicle cycle. We also evaluated the impact of both typical and atypical antipsychotics on MK-801-induced proteome changes. Interestingly, the atypical antipsychotic clozapine showed a more significant capacity to counteract the protein alterations induced by NMDAr hypofunction than haloperidol. Finally, using our dataset, we identified potential modulators of the MK-801-induced proteome changes, which may be considered promising targets to treat NMDAr hypofunction in schizophrenia. This dataset is publicly available and may be helpful in further studies aimed at evaluating the effects of MK-801 and antipsychotics in the human brain.     

Post-mortem changes of neurotransmitter concentrations in the rat brain regions

Using High Performance Liquid Chromatography coupled with electrochemical detection the post-mortem stability of noradrenaline (NA), dopamine (DA), serotonin (5-HT) and 5-hydroxy indole acetic acid (5-HIAA) were examined in the rat hypothalamus, amygdala, cerebral cortex, cerebellum and corpus striatum over an 8 hour time period. Changes in concentrations of the different neurotransmitters were less than it might be expected. The significant changes were: a. A fall in NA levels in the cerebral cortex by 4 hours and in the hypothalamus at 8 hours. b. A reduction in DA concentrations in the corpus striatum at 8 hours but a two fold rise of levels in the hypothalamus at 1 and 2 hours. c. A four-fold increase in 5-HT concentrations in the amygdala throughout the 8 hours studied. The results indicate that for comparative studies on post-mortem brain tissue correction factors should be employed to take into account differential changes in the concentrations of the various neurotransmitters.     

Immunohistochemical changes in vulnerable rat brain regions after reversible global brain ischaemia

Human global ischaemia was simulated in adult rats by inducing 20 min brain ischaemia and 60 min post-ischaemic recirculation. Immunohistochemical expression of MMP-9, TIMP-3, Bax and Bcl-2, and DNA fragmentation (with the TUNEL reaction) were investigated. The morphological data showed different neuronal responses in the hippocampus compared with the cerebral and cerebellar cortices. MMP-9 immunoreactivity was different in the hippocampus, particularly in dentate gyrus and the CA1 region, compared with these cortices. Negative TIMP-3 staining in ischaemic hippocampal neurons may indicate a loss of its inhibitory activity on MMP-9 that could enhance cell death. Bcl-2 down regulation, Bax positivity and TUNEL+ type II cells in the dentate gyrus granular layer could be responsible for induction of apoptotic death in CA1 hippocampal pyramidal cells via loss of fibre input. Results suggest differential behaviours of neural cells after 60 min reperfusion.     

The bombesin/gastrin releasing peptide receptor antagonist RC-3095 blocks apomorphine but not MK-801-induced stereotypy in mice

Bombesin (BN)-like peptides might be involved in the pathogenesis of neuropsychiatric disorders such as schizophrenia. Stereotyped behaviors induced by the dopamine receptor agonist apomorphine or the N-methyl-D-aspartate glutamate receptor antagonist dizocilpine (MK-801) in rodents have been proposed as animal models of schizophrenic psychosis. In the present study we evaluated the effects of the BN/gastrin-releasing peptide receptor (GRP) antagonist (D-Tpi6, Leu13 psi[CH2NH]-Leu14) bombesin (6-14) (RC-3095) on apomorphine and MK-801-induced stereotyped behavior in mice. An intraperitoneal (i.p.) injection of RC-3095 (1.0, 10.0 or 100.0 mg/kg) blocked apomorphine-induced stereotypy. The inhibitory effect of RC-3095 on apomorhine-induced stereotypy was similar to that induced by haloperidol (0.5 mg/kg). RC-3095 did not affect stereotyped behavior induced by MK-801 (0.5 mg/kg). The results provide the first evidence that BN/GRP receptor antagonism blocks stereotyped behavior induced by a dopamine agonist. Together with previous evidence, the present study indicates that the BN/GRP receptor can be considered a drug target in the investigation of potential new agents for treating neuropsychiatric disorders.     

MK-801 changes the role of glutamic acid on modulation of algesia in nucleus accumbens

Dizocilpine maleate (MK-801) causes the blockage of the glutamic acid (Glu) receptors in the central nervous system that are involved in pain transmission. However, the mechanism of action of MK-801 in pain-related neurons is not clear, and it is still unknown whether Glu is involved in the modulation of this processing. This study examines the effect of MK-801, Glu on the pain-evoked response of pain-excitation neurons (PENs) and pain-inhibition neurons (PINs) in the nucleus accumbens (NAc) of rats. The trains of electric impulses applied to the sciatic nerve were used as noxious stimulation. The electrical activities of PENs or PINs in NAc were recorded by a glass microelectrode. Our results revealed that the lateral ventricle injection of Glu increased the discharged frequency and shortened the discharged latency of PEN, and decreased the discharged frequency and prolonged the discharged inhibitory duration (ID) of PIN in NAc of rats evoked by the noxious stimulation, while intra-NAc administration of MK-801 produced the opposite response. On the basis of above findings we can deduce that Glu, MK-801 and N-methyl-d-aspartate (NMDA) receptor are involved in the modulation of nociceptive information transmission in NAc.     

Quinolinic acid but not MK-801 protects the dopaminergic system from 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine-induced toxicity in goldfish retina

The toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, intravitreally injected in goldfish eye, involves interplexiform retinal neurons and depletes tyrosine hydroxylase immunoreactivity and dopamine levels. This induced neurotoxicity was prevented by the concomitant administration in nontoxic doses (10 μg) of quinolinic acid, an endogenous structural analogue of N-methyl -aspartate with excitotoxic properties. Quinolinic acid is ineffective on the retinal degeneration induced by 1-methyl-4- phenylpyridinium ion. This fact suggests that quinolinic acid inhibits the MAO-B oxidation of 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine. MK-801, a noncompetitive antagonist of glutamate NMDA-receptors, exerts partial protective effects on MPTP-induced delayed toxicity in mammals. In the goldfish eye, MK-801, injected in low concentration, and in conjunction with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or 1-methyl-4-phenylpyridinium ion, did not prevent retinal neurodegeneration. Ten μg of MK-801 alone did not affect retinal neurons, while a higher concentration (20 μg) causes the chromatolysis of some photoreceptor nuclei.     

Age-dependent changes of nucleic acid labeling in different rat brain regions

The effects of aging on in vivo DNA and RNA labeling and on RNA content in various brain regions of 4-, 12-, and 24-month-old rats were investigated. No difference in [methyl-¹⁴C]thymidine incorporation into DNA of cerebral cortex and cerebelllum during aging was observed.The ratio of RNA/DNA content significantly decreased from 4 to 24 months of age in cerebral cortex, cerebellum and striatum. RNA labeling decreased by 15% in cerebral cortex of 24-month-old animals while in the other brain areas examined (cerebellum, hippocampus, hypothalamus, brainstem, striatum) did not change during aging.In the cerebral cortex, the ratio of the specific radioactivity of microsomal RNA to that of nuclear RNA, determined by in vivo experiments, was not affected by the aging process. A significant decrease of total, poly(A)+ RNA and poly(A)- RNA content was observed in the same brain area of 24-month-old rats compared to 4-month-old ones. Moreover, densitometric and radioactivity patterns obtained by gel electrophoresis of labeled RNA after in vitro experiments (tissue slices of cerebral cortex) showed a different ribosomal RNA processing during aging. In vivo chronic treatment with CDP-choline was able to increase RNA labeling in corpus striatum of 24-month-old animals.     

Translocation of AIF in the human and rat striatum following protracted haloperidol, but not clozapine treatment

Loss of mitochondrial membrane integrity and consequent release of apoptogenic factors may be involved in mediating striatal neurodegeneration after prolonged treatment with the typical antipsychotic drug haloperidol. Apoptosis-inducing factor (AIF), an intramitochondrial protein, may have a large influence on mediating haloperidol-induced striatal neuron destruction. Translocation of this protein from mitochondria to the nucleus promotes cell death independently of the caspase cascade. To examine how AIF may contribute to haloperidol-induced apoptosis, AIF translocation was observed in three haloperidol treatment paradigms. SH-SY5Y cells were treated with both haloperidol and clozapine and examined for AIF immunofluorescence. Immunohistochemistry was also performed on human striatal sections obtained from the Stanley Foundation Neuropathology Consortium and on rat brain sections following 28 days of antipsychotic drug treatment. In the cellular model haloperidol, but not clozapine treatment increased the nuclear AIF immunofluorescent signal and decreased cell viability. Corollary to these findings, striatal sections from patients who had taken haloperidol and rats who were administered haloperidol both had an elevated nuclear AIF signal. The results provide novel evidence implicating the involvement of AIF in haloperidol-associated apoptosis and its relevance to the development of typical antipsychotic drug-related adverse effects such as tardive dyskinesia.     

Traumatic brain injury and the effects of diazepam,diltiazem, and MK-801 on GABA-A receptor subunit expression in rat hippocampus

Background  

Excitatory amino acid release and subsequent biochemical cascades following traumatic brain injury (TBI) have been well documented, especially glutamate-related excitotoxicity. The effects of TBI on the essential functions of inhibitory GABA-A receptors, however, are poorly understood.     

Effects of chronic hypoxia on MK-801-induced changes in the acute hypoxic ventilatory response.

Chronic hypoxia increases the sensitivity of the central nervous system to afferent input from carotid body chemoreceptors. We hypothesized that this process involves N-methyl-D-aspartate (NMDA) receptor-mediated mechanisms and predicted that chronic hypoxia would change the effect of the NMDA receptor blocker dizocilpine (MK-801) on the poikilocapnic hypoxic ventilatory response (HVR). Male Sprague-Dawley rats were studied before and after acclimatization to hypoxia (70 Torr inspiratory Po(2) for 9 days). We measured ventilation (VI) and the HVR before and after systemic MK-801 treatment (3 mg/kg ip). MK-801 resulted in a constant respiratory frequency (approximately 175 min(-1)) during acute exposure to 10% and 30% O(2) before and after acclimatization. MK-801 had no effect on tidal volume (VT) before acclimatization, but it significantly decreased Vt when the animals were breathing 10% O(2) after acclimatization. The net effect of MK-801 was to eliminate the O(2) sensitivity of Vi before (via changes in respiratory frequency) and after (via changes in VT) acclimatization. Hence, chronic hypoxia altered the effect of MK-801 on the acute HVR, primarily because of increased effects on Vt. This indicates that changes in NMDA receptor-mediated neurotransmission may be involved in ventilatory acclimatization to hypoxia. However, further experiments are necessary to determine the precise location of such plasticity in the central nervous system.     

Tissue levels of haloperidol by radioreceptor assay and behavioral effects of haloperidol in the rat

A radioreceptor assay verified by independent biochemical methods was used to evaluate tissue levels of neuroleptic activity in serum and brain extracts after injections of haloperidol in the rat. The assay detected activity between doses of 0.1 and 10 mg/kg at times between 0.25 and 12 hrs. Tissue levels in blood and brain were highly correlated and corresponded well with a behavioral test of catalepsy at one hour after drug administration. This relationship between brain levels and behavior persisted but changed quantitatively over time.