Regulation of Glutamate Receptors: Implication in Mental Disorders
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Prefrontal cortex (PFC) is the major brain area controlling cognitive and executive functions such as working memory, attention and decision-making. PFC dysfunction has been implicated in several mental disorders such as schizophrenia, attention-deficit hyperactivity disorder (ADHD) and major depressive disorder (MDD). Accordingly, PFC is one of the targets by pharmacological agents used for the treatment of mental disorders. In the PFC, 80 percent of the cellular constituents are the glutamatergic pyramidal neurons. They are innervated by glutamatergic neurons locally or from other brain areas which regulate the excitability of PFC pyramidal neurons correlated to the PFC functions. Glutamatergic transmission is the major excitatory neurotransmission in the brain. Glutamate is released from the presynaptic sites and exerts its action though activating glutamate receptors at postsynaptic terminals. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and N-methyl-D-aspartate (NMDA) receptors are the major glutamate receptors expressed in the PFC. Activation of AMPA receptors (AMPARs) and NMDA receptors (NMDARs) induce membrane depolarization and activation of secondary messages in the neurons. Altered number or function of glutamate receptors at synapse are related to altered synaptic transmission, which are associated with etiology of mental disorders. Despite considerable studies on investigating glutamate receptors in the brain, the relationship between the regulation of glutamate receptors and the actions of certain pharmacological agents is not fully understood. The studies described here investigate the regulation of glutamatergic transmission in PFC by different pharmacological agents. We aimed at understanding the molecular and cellular mechanisms underlying the actions of specific pharmacological agents related to psychiatric disorders. (1) The regulation of surface NMDARs by a group II metabotropic glutamate receptor (group II mGluR) agonist. The group II mGluRs have emerged as the new drug targets for the schizophrenia treatment. To understand the potential mechanisms underlying the antipsychotic effects of group II mGluRs agonists, we examined their impact on NMDARs, since NMDAR hypofunction has been implicated in the etiology of schizophrenia. We found that activation of group II mGluRs by APDC, a selective agonist, caused a significant enhancement of NMDAR-mediated currents in cultured cortical pyramidal neurons, which was associated with an increased level of NMDAR surface expression and synaptic localization. Furthermore, we found that the enhancing effect of APDC on NMDAR-mediated currents was abolished when botulinum toxin was delivered into the recorded neurons to disrupt the SNARE complex, a family of proteins involved in vesicle trafficking and membrane fusion. Inhibiting the function of two key SNARE proteins, SNAP-25 and syntaxin 4, also eliminated the effect of APDC on NMDAR-mediated currents. Moreover, application of APDC increased the activity of Rab4, a small Rab GTPase mediating fast vesicle recycling from early endosomes to the plasma membrane, and enhanced the interaction between syntaxin 4 and Rab4. Knockdown of Rab4 or expression of dominant-negative Rab4 attenuated the effect of APDC on NMDAR currents. Taken together, these results have identified key molecules involved in the group II mGluR-induced potentiation of NMDAR exocytosis and function, which may underlie the antipsychotic effects of group II mGluR agonist. (2) The dose-dependent effects of methylphenidate (MPH) on glutamate receptors in PFC and animal behavior. MPH is a prescribed psychostimulant drug widely used for the treatment of attention-deficit hyperactivity disorder (ADHD) in young people. While MPH produces the effects of increasing alertness and improving attention, its misuse and abuse has been associated with an increased risk of aggression and psychosis. To achieve therapeutic benefits and minimal side-effects, it is suggested that the dosage of MPH should be titrated to an optimal level. In this study, we determined the molecular mechanisms underlying the complex actions of MPH at different dosages. We found that a single administration of low-dose (0.5 mg/kg) MPH facilitated the PFC-dependent cognitive processes including temporal order recognition memory (TORM) and attentional-set shifting without altering locomotor activity in adolescent (4-week-old) rats. Conversely, animals injected with high-dose (10 mg/kg) MPH exhibited significantly elevated locomotor activity and impaired TORM and attentional-set shifting. At the cellular level, we found that low-dose MPH selectively potentiated NMDAR-mediated excitatory postsynaptic currents (EPSCs) while the high-dose MPH suppressed both NMDAR- and AMPAR-EPSCs on pyramidal neurons in the rat PFC. The dual effects of MPH on EPSCs were associated with bi-directional regulation on the surface level of glutamate receptor subunits. Moreover, we found that the enhancing effect of low-dose MPH on NMDAR-EPSCs was attributed to the inhibition of norepinephrine reuptake and the activation of adrenergic receptors. As to the downstream moleculars in the regulatory pathway, inhibiting protein kinase C (PKC) and SNAP-25, a key SNARE protein involved in the exocytosis of NMDARs, blocked the increase of NMDAR-EPSCs by low-dose MPH. Taken together, our results have provided potential mechanisms underlying the cognitive enhancing effects of low-dose MPH, as well as the psychosis inducing effects of high-dose MPH. (3) The rescue of the impaired cognitive function and repressed glutamatergic transmission by low-dose methylphenidate in restraint stressed animals. Previous studies have shown that temporal order recognition memory (TORM) and NMDAR function are impaired in the adolescent rats exposed to repeatedly restraint stress (2 hr for 7 days). In the present study, we found that a single administration of low-dose MPH (0.5 mg/kg) effectively restored the impaired TORM in the restraint stressed animals via a SNAP-25-dependent mechanism. Furthermore, the repressed NMDAR-EPSC on the PFC pyramidal neurons in the animals exposed to the restraint stress was completely restored by low-dose MPH. Inhibiting the function of endogenous SNAP-25 blocked the rescue of low-dose MPH on NMDAR-EPSCs. These results suggest that low-dose MPH effectively rescues the restraint stress-induced impairment in TORM and NMDAR function in adolescent rats, implying the ability of MPH to combat stress. (4) The involvement of DNA methylation in the severe stress-induced long-lasting impairment of glutamatergic transmission and animal behavior. The severity and duration of stressor greatly affects the outcomes of stress. While mild chronic stress could be adapted physiologically and behaviorally, severe chronic stress causes long-lasting detrimental effects and acts as a trigger for several mental disorders such as major depressive disorder. Our animal behavioral tasks showed that severe stress (6 hr per day for 7 days) exerted a long-lasting impairment in the cognitive function and locomotor activity in the adolescent rats, which sustained three weeks after withdrawal from stress. Correspondingly, the glutamatergic transmission in rat PFC was remarkably repressed in the animals exposed to severe stress, which lasted at least for one week after stress. Different than the mild stressed animals, we found that the mRNA level of DNA methyltransferases 3a (Dnmt3a) and 3b (Dnmt3b) were significantly reduced in the severe stressed animals. Since DNA methylation is associated with repressed gene transcription and implicated in the activity-dependent long-lasting modification of synaptic plasticity and brain function, we further examined the involvement of DNA methylation in the actions of severe stress. 5-AzaCytosine (5AzaC) and RG108, selective DNA methyltransferases (DNMT) inhibitors, were applied in the naïve animals to examine whether they mimicked the effects of severe stress. Behavioral tasks showed that the recognition memory was significantly impaired in the animals treated with DNMT inhibitors. Electrophysiological evidence showed that the NMDAR-EPSCs and AMPA EPSCs on the PFC pyramidal neurons were substantially decreased in animals treated with 5AzaC and RG108. These results suggested that DNA methylation may mediate the severe stress-induced long-lasting deficits in the cognitive function and glutamatergic transmission in PFC. Further studies need to be done to confirm the findings and investigate the underlying molecular mechanisms. Given the importance of glutamatergic transmission in the PFC, our studies identified the glutamate receptors as the target of group II mGluRs agonist and MPH, essential for their therapeutic actions. In addition, our preliminary data suggest that the impaired glutamatergic transmission and cognitive functions induced by severe stress may though an epigenetic mechanism. These studies broaden our understanding of the actions of pharmacological agents and provide insights into the therapeutic strategy and novel drug discovery.