Cocaine enhances NMDA receptor-mediated currents in ventral tegmental area cells via dopamine D5 receptor-dependent redistribution of NMDA receptors.
ABSTRACT: Cocaine-induced plasticity of glutamatergic synaptic transmission in the ventral tegmental area (VTA) plays an important role in brain adaptations that promote addictive behaviors. However, the mechanisms responsible for triggering these synaptic changes are unknown. Here, we examined the effects of acute cocaine application on glutamatergic synaptic transmission in rat midbrain slices. Cocaine caused a delayed increase in NMDA receptor (NMDAR)-mediated synaptic currents in putative VTA dopamine (DA) cells. This effect was mimicked by a specific DA reuptake inhibitor and by a DA D1/D5 receptor agonist. The effect of cocaine was blocked by a DA D1/D5 receptor antagonist as well as by inhibitors of the cAMP/cAMP-dependent protein kinase A (PKA) pathway. Furthermore, biochemical analysis showed an increase in the immunoreactivity of the NMDAR subunits NR1 and NR2B and their redistribution to the synaptic membranes in VTA neurons. Accordingly, NMDAR-mediated EPSC decay time kinetics were significantly slower after cocaine, suggesting an increased number of NR2B-containing NMDARs. Finally, pharmacological analysis indicates that NR2B subunits might be incorporated in triheteromeric NR1/NR2A/NR2B complexes rather than in "pure" NR1/NR2B NMDA receptors. Together, our data suggest that acute cocaine increases NMDAR function in the VTA via activation of the cAMP/PKA pathway mediated by a DA D5-like receptor, leading to the insertion of NR2B-containing NMDARs in the membrane. These results provide a potential mechanism by which acute cocaine promotes synaptic plasticity of VTA neurons, which could ultimately lead to the development of addictive behaviors.
Project description:Exposure to drug-associated cues evokes drug-seeking behavior and is regarded as a major cause of relapse. Cues evoke burst firing of ventral tegmental area (VTA) dopamine (DA) neurons and phasic DA release in the nucleus accumbens (NAc). Cholinergic and glutamatergic input to the VTA is suggested to gate phasic DA activity. However, the role of VTA cholinergic and glutamatergic receptors in regulating phasic dopamine release and cue-induced drug-seeking in cocaine experienced subjects is not known. In male Sprague-Dawley rats, we found that VTA inactivation strongly inhibited, while VTA stimulation promoted, cocaine-seeking behavior during early withdrawal. Blockade of phasic activated D1 receptors in the NAc core also strongly inhibited cue-induced cocaine-seeking--suggesting an important role of phasic DA activity in the VTA to NAc core circuit. Next, we examined the role of VTA acetylcholine receptors (AChRs) and N-methyl-D-aspartate receptors (NMDARs) in regulating both NAc core phasic DA release and cue-induced cocaine-seeking. In cocaine naïve subjects, VTA infusion of the nicotinic acetylcholine receptor (AChR) antagonist mecamylamine, the muscarinic AChR antagonist scopolamine, or the NMDAR antagonist AP-5, led to robust attenuation of phasic DA release in the NAc core. During early cocaine withdrawal, VTA infusion of AP-5 had limited effects on NAc phasic DA release and cue-induced cocaine-seeking while VTA infusion of mecamylamine or scopolamine robustly inhibited both phasic DA release and cocaine-seeking. The results demonstrate that VTA AChRs, but not NMDARs, strongly regulate cue-induced cocaine-seeking and phasic DA release during early cocaine withdrawal.
Project description:NMDA type glutamate receptors (NMDARs) are best known for their role in synaptogenesis and synaptic plasticity. Much less is known about their developmental role before neurons form synapses. We report here that VEGF, which promotes migration of granule cells (GCs) during postnatal cerebellar development, enhances NMDAR-mediated currents and Ca(2+) influx in immature GCs before synapse formation. The VEGF receptor Flk1 forms a complex with the NMDAR subunits NR1 and NR2B. In response to VEGF, the number of Flk1/NR2B coclusters on the cell surface increases. Stimulation of Flk1 by VEGF activates Src-family kinases, which increases tyrosine phosphorylation of NR2B. Inhibition of Src-family kinases abolishes the VEGF-dependent NR2B phosphorylation and amplification of NMDAR-mediated currents and Ca(2+) influx in GCs. These findings identify VEGF as a modulator of NMDARs before synapse formation and highlight a link between an activity-independent neurovascular guidance cue (VEGF) and an activity-regulated neurotransmitter receptor (NMDAR).
Project description:NMDA receptors (NMDARs) are tetrameric protein complexes usually comprising two NR1 and two NR2 subunits. Different combinations of four potential NR2 subunits (NR2A-D) confer diversity in developmental expression, subsynaptic localization, and functional characteristics, including affinity for neurotransmitter. NR2B-containing NMDARs, for example, exhibit relatively high affinity both for glutamate and the coagonist glycine. Although multiple NMDAR subtypes can colocalize at individual synapses, particular subtypes often mediate inputs from distinct functional pathways. In retinal ganglion cells (RGCs), NMDARs contribute to synaptic responses elicited by light stimulus onset ("ON") and offset ("OFF"), but roles for particular NMDAR subtypes, and potential segregation between the ON and OFF pathways, have not been explored. Moreover, elements in the retinal circuitry release two different NMDAR coagonists, glycine and d-serine, but the effects of endogenous coagonist release on the relative contribution of different NMDAR subtypes are unclear. Here, we show that coagonist release within the retina modulates the relative contribution of different NMDARs in the ON pathway of the rat retina. By pharmacologically stimulating functional pathways independently in acute slices and recording synaptic responses in RGCs, we show that ON inputs, but not OFF inputs, are mediated in part by NMDARs exhibiting NR2B-like pharmacology. Furthermore, suppressing release of NMDAR coagonist reduces NMDAR activation at ON synapses and increases the relative contribution of these putative NR2B-containing receptors. These results demonstrate direct evidence for evoked coagonist release onto NMDARs and indicate that modulating coagonist release may regulate the relative activation of different NMDAR subtypes in the ON pathway.
Project description:Synaptic plasticity is induced by the influx of calcium ions (Ca2+) through N-methyl-D-aspartate receptors (NMDARs), and the direction and strength of the response depend on the frequency of the synaptic inputs. Recent studies have shown that the direction of synaptic plasticity is also governed by two distinct NMDAR subtypes (NR1/NR2A, NR1/NR2B). How are the different types of regulation (frequency-dependent and receptor-specific) processed simultaneously? To clarify the molecular basis of this dual dependence of synaptic plasticity, we have developed a mathematical model of spatial Ca2+ signaling in a dendritic spine. Our simulations revealed that calmodulin (CaM) activation in the vicinity of NMDARs is strongly affected by the diffusion coefficient of CaM itself, and that this 'local CaM diffusion system' works as a dual decoder of both the frequency of Ca2+ influxes and their postsynaptic current shapes, generated by two NMDAR subtypes, implying that spatial factors may underlie the complicated regulation scheme of synaptic plasticity.
Project description:NMDA receptor (NMDAR)-mediated excitatory synaptic transmission plays a critical role in synaptic plasticity and memory formation, whereas its dysfunction may underlie neuropsychiatric and neurodegenerative diseases. The neuroactive steroid pregnenolone sulfate (PS) acts as a cognitive enhancer in impaired animals, augments LTP in hippocampal slices by enhancing NMDAR activity, and may participate in the reduction of schizophrenia's negative symptoms by systemic pregnenolone. We report that the effects of PS on NMDAR function are diverse, varying with subunit composition and NR1 splice variant. While PS potentiates NR1-1a/NR2B receptors through a critical steroid modulatory domain in NR2B that also modulates tonic proton inhibition, potentiation of the NMDA response is not dependent upon relief of such inhibition, a finding that distinguishes it from spermine. In contrast, the presence of an NR2A subunit confers enhanced PS-potentiation at reduced pH, suggesting that it may indeed act like spermine does at NR2B-containing receptors. Additional tuning of the NMDAR response by PS comes via the N-terminal exon-5 splicing insert of NR1-1b, which regulates the magnitude of proton-dependent PS potentiation. For NR2C- and NR2D-containing receptors, negative modulation at NR2C receptors is pH-independent (like NR2B) while negative modulation at NR2D receptors is pH-dependent (like NR2A). Taken together, PS displays a rich modulatory repertoire that takes advantage of the structural diversity of NMDARs in the CNS. The differential pH sensitivity of NMDAR isoforms to PS modulation may be especially important given the emerging role of proton sensors to both learning and memory, as well as brain injury.
Project description:Blockade of N-methyl-D-aspartate receptors (NMDARs) produces behavior in healthy people that is similar to the psychotic symptoms and cognitive deficits of schizophrenia and can exacerbate symptoms in people with schizophrenia. However, an endogenous brain disruption of NMDARs has not been clearly established in schizophrenia. We measured mRNA transcripts for five NMDAR subunit mRNAs and protein for the NR1 subunit in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia and control (n=74) brains. Five NMDAR single-nucleotide polymorphisms (SNPs) previously associated with schizophrenia were tested for association with NMDAR mRNAs in postmortem brain and for association with cognitive ability in an antemortem cohort of 101 healthy controls and 48 people with schizophrenia. The NR1 subunit (mRNA and protein) and NR2C mRNA were decreased in postmortem brain from people with schizophrenia (P=0.004, P=0.01 and P=0.01, respectively). In the antemortem cohort, the minor allele of NR2B rs1805502 (T5988C) was associated with significantly lower reasoning ability in schizophrenia. In the postmortem brain, the NR2B rs1805502 (T5988C) C allele was associated with reduced expression of NR1 mRNA and protein in schizophrenia. Reduction in NR1 and NR2C in the DLPFC of people with schizophrenia may lead to altered NMDAR stoichiometry and provides compelling evidence for an endogenous NMDAR deficit in schizophrenia. Genetic variation in the NR2B gene predicts reduced levels of the obligatory NR1 subunit, suggesting a novel mechanism by which the NR2B SNP may negatively influence other NMDAR subunit expression and reasoning ability in schizophrenia.
Project description:NMDA-type glutamate receptors (NMDARs) are major contributors to long-term potentiation (LTP), a form of synaptic plasticity implicated in the process of learning and memory. These receptors consist of calcium-permeating NR1 and multiple regulatory NR2 subunits. A majority of studies show that both NR2A and NR2B-containing NMDARs can contribute to LTP, but their unique contributions to this form of synaptic plasticity remain poorly understood.In this study, we show that NR2A and NR2B-containing receptors promote LTP differently in the CA1 hippocampus of 1-month old mice, with the NR2A receptors functioning through Ras-GRF2 and its downstream effector, Erk Map kinase, and NR2B receptors functioning independently of these signaling molecules.This study demonstrates that NR2A-, but not NR2B, containing NMDA receptors induce LTP in pyramidal neurons of the CA1 hippocampus from 1 month old mice through Ras-GRF2 and Erk. This difference add new significance to the observation that the relative levels of these NMDAR subtypes is regulated in neurons, such that NR2A-containing receptors become more prominent late in postnatal development, after sensory experience and synaptic activity.
Project description:The NR2 subunit composition of NMDA receptors (NMDARs) varies during development, and this change is important in NMDAR-dependent signaling. In particular, synaptic NMDAR switch from containing mostly NR2B subunit to a mixture of NR2B and NR2A subunits. The pathways by which neurons differentially traffic NR2A- and NR2B-containing NMDARs are poorly understood. Using single-particle and -molecule approaches and specific antibodies directed against NR2A and NR2B extracellular epitopes, we investigated the surface mobility of native NR2A and NR2B subunits at the surface of cultured neurons. The surface mobility of NMDARs depends on the NR2 subunit subtype, with NR2A-containing NMDARs being more stable than NR2B-containing ones, and NR2A subunit overexpression stabilizes surface NR2B-containing NMDARs. The developmental change in the synaptic surface content of NR2A and NR2B subunits was correlated with a developmental change in the time spent by the subunits within synapses. This suggests that the switch in synaptic NMDAR subtypes depends on the regulation of the receptor surface trafficking.
Project description:N-methyl-d-aspartate receptors (NMDARs) play an important role in many aspects of nervous system function such as synaptic plasticity and neuronal development. NMDARs are heteromers consisting of an obligate NR1 and most commonly one or two kinds of NR2 subunits. While the receptors have been well characterized in some vertebrate and invertebrate systems, information about NMDARs in Xenopus laevis brain is incomplete. Here we provide biochemical evidence that the NR1, NR2A and NR2B subunits of NMDARs are expressed in the central nervous system of X. laevis tadpoles. The NR1-4a/b splice variants appear to be the predominant isoforms while the NR1-3a/b variants appear to be expressed at low levels. We cloned the X. laevis NR2A and NR2B subunits and provide a detailed annotation of their functional domains in comparison with NR2A and NR2B proteins from 10 and 13 other species, respectively. Both NR2A and NR2B proteins are remarkably well conserved between species, consistent with the importance of NMDARs in nervous system function.
Project description:<h4>Background</h4>Zinc distributes widely in the central nervous system, especially in the hippocampus, amygdala and cortex. The dynamic balance of zinc is critical for neuronal functions. Zinc modulates the activity of N-methyl-D-aspartate receptors (NMDARs) through the direct inhibition and various intracellular signaling pathways. Abnormal NMDAR activities have been implicated in the aetiology of many brain diseases. Sustained zinc accumulation in the extracellular fluid is known to link to pathological conditions. However, the mechanism linking this chronic zinc exposure and NMDAR dysfunction is poorly understood.<h4>Methodology/principal findings</h4>We reported that chronic zinc exposure reduced the numbers of NR1 and NR2A clusters in cultured hippocampal pyramidal neurons. Whole-cell and synaptic NR2A-mediated currents also decreased. By contrast, zinc did not affect NR2B, suggesting that chronic zinc exposure specifically influences NR2A-containg NMDARs. Surface biotinylation indicated that zinc exposure attenuated the membrane expression of NR1 and NR2A, which might arise from to the dissociation of the NR2A-PSD-95-Src complex.<h4>Conclusions</h4>Chronic zinc exposure perturbs the interaction of NR2A to PSD-95 and causes the disorder of NMDARs in hippocampal neurons, suggesting a novel action of zinc distinct from its acute effects on NMDAR activity.