Group â…¡ Metabotropic Glutamate Receptor Agonists Ameliorate MK-801-induced NMDA Receptor Dysfunction In Adult Rat Prefrontal Cortex

Objective:The prefrontal cortex(PFC) is a complex brain structure composed of heterogeneous cell types with distinct morphologies and functions.The inhibitory GABAergic system consists of many different subclasses of interneurons,each containing different calciumbinding protein(CaBP),such as parvalbumin(PV).Previous studies have indicated that parvalbumin-containing interneurons are selectively damaged in schizophrenia.This creates technical challenges for analyzing the relevant molecular mechanisms,because current in vivo biochemical techniques rely on tissue homogenization and it is unavoidable to miss the alteration of genes expressions in specific cell population.To achieve molecular analysis of morphologically and phenotypically identified cells, methods for obtaining specific cell groups have been developed.Laser Microdissection (LMD) is relatively new technique with the capability of selectively picking up a subpopulation of neurons under microscopic visualization.Real-time PCR is a powerful method for quantification of gene expression.The combination of them makes it possible to analyze genes expression in parvalbumin-containing interneurons.Although parvalbumin-containing interneurons were reported to be more sensitive to NMDA receptor antagonists,whether these cells express NMDA receptor subunits is still unknown.Several distinct NMDAR subtypes have now been identified in central neurons, differing in their sensitivity to endogenous and exogenous ligands,permeation and block by divalent ions,kinetic properties,and interaction with intracellular proteins:the ubiquitously expressed NR1 subunit;a family of four distinct NR2 subunits(NR2A, NR2B,NR2C,NR2D);and NR3 subunits.In order to study the distinct properties of parvalbumin-containing interneurons,we have designed a combination technique composing of LMD,aRNA amplification and real-time PCR to examine the NMDA receptor subunits expression in these cells versus the PFC tissue.Methods: 1.NovaRed rapid staining 2.Laser Microdissection(LMD) 3.RNA extraction 4.Reverse transcription polymerase chain reaction(RT-PCR) 5.anti-sense RNA (aRNA) amplification 6.Real-time PCR 7.Immunofluorescence 8.Western blottingResults:The composition of NMDA receptor subunits in PV-ir interneurons differs from the PFC.All the designed primers of NMDA receptor subunits are proved to be with high specificity and sensitivity.The RNA isolated from fresh PFC tissue and PV-ir interneurons obtained by LMD is with high quality.All of the NR1,NR2 subunits are expressed in the PFC.The normalized mRNA levels of NMDA receptor subunits are: NR 1,7.76±1.78;NR2A,1.92±0.66;NR2B,31.7±3.91;NR2C,1.89±0.45;NR2D, 1.52±0.41.The mRNA expression of NR2B subunits is much higher than NR2A in the PFC(p=0.0001).The protein levels of NR2B in the pyramidal neurons(8.19±0.41) in the PFC is also significantly higher than the levels of NR2A subunits(4.57±0.23). However,NR1,NR2A,NR2B,NR2C,but no NR2D,are expressed by the PV-ir interneurons.The normalized mRNA levels of these subunits are:NR1,35.7±11.4; NR2A,77.1±28.3;NR2B,10.7±4.26;NR2C,27.3±3.62.Comparably,the NR2B level is significantly lower than NR2A in the PV-ir interneurons(p=0.031).In consistent,the protein levels of NR2B(4.07±0.64) in the PV-ir interneurons is also lower than NR2A level(5.33±0.27).Thus the NR2A/NR2B ratio in PV-ir interneurons is significantly higher compared with that in the PFC tissue(p<0.01).Conclusion:A combination of techniques is introduced to examine the expression of mRNA for NMDA receptor subunits in PV-ir interneurons in rat PFC.It has made it possible to study the characteristics of mRNA expression in a specific cell population.The ability to analyze a single cell type is an important distinction from global and regional assessments of mRNA expression,representing progress in gene detection from tissue to cellular level.NMDA receptor subunits(NR1,NR2A,NR2B,NR2C,but not NR2D) are expressed in PV-ir interneurons,with a significantly high proportion of NR2A and low NR2B,suggesting the unique property of this specific cell population. Objective:Based on studies showing that non-competitive antagonists of NMDA receptors, such as MK-801,induce a psychosis resembling both the positive and negative symptoms, the "NMDA receptor hypofunction" hypothesis emerged in the early 1980s as an alternative to the prevailing theory of altered dopamine neurotransmission.NMDA receptor antagonists were widely used in schizophrenia animal model,but controversial results had been reported in the alteration of NMDA receptor expression,which is inconsistent with the hypothesized hypofunction.Furthermore,the drugs functioning directly to activate NMDA receptor did not succeed in eliminating the schizophrenic symptoms.In vitro studies indicated that the interneurons,especially parvaibumin-immunoreactive (PV-ir) cells,were more sensitive to NMDA receptor antagonists than pyramidal neurons,and were selectively disrupted in schizophrenia.This raised the current theory that PV-ir interneurons may function as the sensor for NMDA receptor in the pathological mechanism,and the inhibition of NMDA channels by antagonists might reduce inhibitory output and depolarize the excitatory glutamatergic pyramidal neurons. Therefore,pyramidal cell activity would increase and release large amount of glutamate. Prolonged overactivity of pyramidal neurons could have deleterious consequences,cause cell swelling and signs of cellular stress.This intriguing hypothesis,however,has not been tested..Considering the possible excitotoxicity of overactivated NMDA receptor,we hypothesize that different dosages of MK-801 may induce distinct,or even opposite, alteration of NMDA receptor expression.Regardless of the level in the PFC,how the receptor in PV-ir interneurons is affected? Are the changes consistent with those in the PFC? Therefore,we applied subchronic administration with MK-801 at different dosages and examine the alterations of NMDA receptor subunits mRNA and protein in PV-ir interneuron versus the PFC to study the mechanism of schizophrenia at the cellular level.Methods: 1.Subchronic administration of MK-801 at different dosages in adult female rats 2. Combined techniques to examine the mRNA expression of NMDA receptor in PV-ir interneurons 3.Immunofluorescence 4.Western blottingResults:MK-801 induces distinct alterations of NMDA receptor subunits in PV-ir interneurons.NMDA receptor expressions in five MK-801-treated groups at different dosages are compared to control group.With the increase of dosage,all 5 subunits in the PFC,including NR1 and NR2A toNR2D,showed inverted-U dose-dependent changes, i.e.,increased at low doses but decreased at high dose,although slight differences existed among the 5 NMDAR subunits examined.Subchronic treatment with MK-801 at doses of 0.01 to 0.1 mg/kg significantly upregulated the mRNA expressions of all subunits in the PFC,with NR1 increased by 1.6- to 2.0-fold,NR2A by as high as 2.3- to 3.9-fold,NR2B by 1.8- to 2.5-fold,NR2C by 1.6- to 3.5-fold,and NR2D by 1.9- to 3.8-fold(P＜0.05 for all subunits).At higher dose of 0.33 mg/kg,all subunits exhibited no significant changes, whereas at a high dose of 1.0 mg/kg,all subunit significantly decreased to almost undetectable levels(P＜0.01 for all) except for NR2D,which showed a 1.9-fold decrease but not statistically different(P=0.149).However,the receptor subunits in PV-ir interneurons respond to MK-801 well in a biphasic pattern.The mRNA expressions of all NMDAR subunits were significant decreased at low doses of 0.01 and 0.033 mg/kg(P＜0.05) except for a slight decrease of NR2C at 0.01 mg/kg(P=0.327) and almost no change of NR1 at 0.1 mg/kg(P=0.909).Similar to the mRNA expressions in PFC tissue, all subunits exhibited no significant changes at 0.33 mg/kg(P＞0.05) but significant decreases at high dose of MK-801(1.0 mg/kg,P＜0.05).These results suggest that PV-containing interneurons in the PFC have a distinct responsiveness to NMDAR antagonism.These alterations are consistent with the protein studies,which is performed by choosing two typical doses,which are 0.033 mg/kg for the opposite regulation and 1.0 mg/kg for the consistent down-regulation of NMDA receptor in the PFC and PV-ir interneurons.Additionally,treatment with MK-801 at the dosage of both 0.033 mg/kg and 1.0 mg/kg significantly decreases the phosphorylation of NR2B(pNR2B tyr1472, 0.634±0.037 for MK-801 at 0.033mg/kg,0.752±0.055 for MK-801 at 1.0 mg/kg,1.0 ±0.04 for control;and pNR2B ser1303,0.601±0.025 for MK-801 at 0.033 mg/kg,0.576±0.035 for MK-801 at 1.0 mg/kg,1.0±0.041 for control)(p＜0.05).Conclusion:Subchronic treatment with MK-801 induces distinct alteration of NMDA receptor in PV-ir interneurons,showing a biphasic pattern of mRNA expression with the increase of MK-801 dosage,while NMDA receptor in the PFC presented with a inverted- U dose-dependent alteration.It suggests that PV-ir interneuron is more sensitive to MK-801,and may functions as a sensor for homeostatic regulation of the PFC.The different regulation of NMDA receptor by different dosage of MK-801 may mimic distinct schizophrenia animal model,and explain why pharmacological intervention,which directly targets on NMDA receptor,does not recover schizophrenic symptoms. Objective:Currently typical antipsychotic drug such as haloperidol and atypical drug such as clozapine(both mainly act as dopamine receptor 2 antagonists) are widely used for treatment of schizophrenia.However,side effects are usually detected and the negative and cognitive symptoms are not significantly affected in many schizophrenics after drug administration.As several lines of evidence implicated NMDA receptor dysfunction in the cognitive deficits of schizophrenia,pharmacological manipulation of the NMDA receptor may be a feasible therapeutic strategy for treatment of these symptoms. Unfortunately studies indicated that D-serine,glycine and D-alanine,which directly activate the NMDA receptors,failed to reverse the deficits of schizophrenia.The study of metabotropic glutamate receptor(mGluR) has changed the classical view of glutamate receptors.Although glutamate is considered as excitatory neurotransmitter,activation of mGluR induces inhibitory effects,and indirectly modulates function of NMDA receptors,mGluR differs from other G protein- coupled receptor by a specific domain connected to N terminal,composed by 550 amino acids.It has 8 different subunits,which are divided into three groups by the sequence,signaling pathway and sensitivity to the agonists.Different groups are located in different brain regions and this brought the opportunity of developing drugs that modify glutamate neurotransmission,because the drugs targeting on specific mGluR subunit,can selectively modulate the function of NMDA receptor in specific region or cell type.Behavioral studies indicated that single injection of mGluRⅡagonist eliminated the symptoms in schizophrenia animal model,but the involved cellular and molecular mechanisms have not been examined.Furthermore,how would mGluRⅡagonists affect the function of NMDA receptor and whether it can reverse MK-801-induced PFC dysfunction are still unknown.Therefore,we examine the alternation of NMDA receptor expression and phosphorylation induced by treatment with mGluRⅡagonists in subchronic treatment with MK-801 animal model.Methods: 1.Schizophrenia animal model by MK-801 administration 2.Western blotting 3. RT-PCRResults:mGluRⅡagonists effectively reversed the MK-801-induced dysfunction of NMDA receptor via GSK-3βsignaling pathway in the PFC.Acute treatment with LY379268 at 0.3mg/kg and 3.0mg/kg significantly increased the expression and phosphorylation of NR2B subunits(pNR2B ser 1303,1.642±0.142 at 0.3 mg/kg,1.453±0.149 at 3.0 mg/kg,1.0±0.147 for control)(p＜0.05).Subchronic treatment with mGluR2/3 agonist LY379268 after MK-801 administration at the corresponding dosages(0.3 mg/kg LY379268 for 0.033 mg/kg MK-801 and 3.0 mg/kg LY379268 for 1.0 mg/kg MK-801) significantly reversed MK-801-induced decrease of pNR2B ser 1303(p＜0.05) to control levels.LY379268 at 0.3 mg/kg recovered MK-801-induced increase of NMDA receptor subunits at 0.033 mg/kg(p＜0.05).Although compared to the level in 1.0 mg/kg MK-801-treated group,NMDA receptor subunits was significantly increased by LY379268 at 3.0 mg/kg(p＜0.05),the expression level is still much lower than that of control group (p＜0.05).Conclusion:mGluRⅡagonists used to be hypothesized to eliminate schizophrenic symptoms through modulating presynaptic glutamate release.However,it is unknown why a glutamatergic release inhibitor would share similar antipsychotic effect with the traditional dopamine receptor 2 antagonists.Our study,for the first time,focused on the postsynaptic effects,indicating that mGluRⅡagonists modulate the function of NMDA receptors and reverse the MK-801-induced PFC dysfunction.