AB1555 Sigma-AldrichAnti-NMDAR2A Antibody

Learned associations between environmental cues and morphine use play an important role in the maintenance and/or relapse of opioid addiction. Although previous studies suggest that context-dependent morphine treatment alters glutamatergic transmission and synaptic plasticity in the hippocampus, their role in morphine conditioned place preference (CPP) and reinstatement remains unknown. We investigated changes in synaptic plasticity and NMDAR expression in the hippocampus after the expression, extinction, and reinstatement of morphine CPP. Here we report that morphine CPP is associated with increased basal synaptic transmission, impaired hippocampal long-term potentiation (LTP), and increased synaptic expression of the NR1 and NR2b NMDAR subunits. Changes in synaptic plasticity, synaptic NR1 and NR2b expression, and morphine CPP were absent when morphine was not paired with a specific context. Furthermore, hippocampal LTP was impaired and synaptic NR2b expression was increased after extinction of morphine CPP, indicating that these alterations in plasticity may be involved in the mechanisms underlying the learning of drug-environment associations. After extinction of morphine CPP, a priming dose of morphine was sufficient to reinstate morphine CPP and was associated with LTP that was indistinguishable from saline control groups. In contrast, morphine CPP extinguished mice that received a saline priming dose did not show CPP and had disrupted hippocampal LTP. Finally, we found that reinstatement of morphine CPP was prevented by the selective blockade of the NR2b subunit in the hippocampus. Together, these data suggest that alterations in synaptic plasticity and glutamatergic transmission play an important role in the reinstatement of morphine CPP.

Loss of sensory input from peripheral organ damage, sensory deprivation, or brain damage can result in adaptive or maladaptive changes in sensory cortex. In previous research, we found that auditory cortical tuning and tonotopy were impaired by cross-modal invasion of visual inputs. Sensory deprivation is typically associated with a loss of inhibition. To determine whether inhibitory plasticity is responsible for this process, we measured pre- and postsynaptic changes in inhibitory connectivity in ferret auditory cortex (AC) after cross-modal plasticity. We found that blocking GABAA receptors increased responsiveness and broadened sound frequency tuning in the cross-modal group more than in the normal group. Furthermore, expression levels of glutamic acid decarboxylase (GAD) protein were increased in the cross-modal group. We also found that blocking inhibition unmasked visual responses of some auditory neurons in cross-modal AC. Overall, our data suggest a role for increased inhibition in reducing the effectiveness of the abnormal visual inputs and argue that decreased inhibition is not responsible for compromised auditory cortical function after cross-modal invasion. Our findings imply that inhibitory plasticity may play a role in reorganizing sensory cortex after cross-modal invasion, suggesting clinical strategies for recovery after brain injury or sensory deprivation.

The influence of the gut microbiota on brain chemistry has been convincingly demonstrated in rodents. In the absence of gut bacteria, the central expression of brain derived neurotropic factor, (BDNF), and N-methyl-d-aspartate receptor (NMDAR) subunits are reduced, whereas, oral probiotics increase brain BDNF, and impart significant anxiolytic effects. We tested whether prebiotic compounds, which increase intrinsic enteric microbiota, also affected brain BDNF and NMDARs. In addition, we examined whether plasma from prebiotic treated rats released BDNF from human SH-SY5Y neuroblastoma cells, to provide an initial indication of mechanism of action. Rats were gavaged with fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) or water for five weeks, prior to measurements of brain BDNF, NMDAR subunits and amino acids associated with glutamate neurotransmission (glutamate, glutamine, and serine and alanine enantiomers). Prebiotics increased hippocampal BDNF and NR1 subunit expression relative to controls. The intake of GOS also increased hippocampal NR2A subunits, and frontal cortex NR1 and d-serine. Prebiotics did not alter glutamate, glutamine, l-serine, l-alanine or d-alanine concentrations in the brain, though GOSfeeding raised plasma d-alanine. Elevated levels of plasma peptide YY (PYY) after GOS intake was observed. Plasma from GOS rats increased the release of BDNF from SH-SY5Y cells, but not in the presence of PYY antisera. The addition of synthetic PYY to SH-SY5Y cell cultures, also elevated BDNF secretion. We conclude that prebiotic-mediated proliferation of gut microbiota in rats, like probiotics, increases brain BDNF expression, possibly through the involvement of gut hormones. The effect of GOS on components of central NMDAR signalling was greater than FOS, and may reflect the proliferative potency of GOS on microbiota. Our data therefore, provide a sound basis to further investigate the utility of prebiotics in the maintenance of brain health and adjunctive treatment of neuropsychiatric disorders.

Protease-activated receptor-1 (PAR1) is an unusual G-protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1-/- mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1-/- mice have deficits in hippocampus-dependent memory. We also show that while PAR1-/- mice have normal baseline synaptic transmission at Schaffer collateral-CA1 synapses, they exhibit severe deficits in N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR-mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR-dependent processes subserving memory formation and synaptic plasticity.

Although alcoholism is a worldwide problem resulting in millions of deaths, only a small percentage of alcohol users become addicted. The specific neural substrates responsible for individual differences in vulnerability to alcohol addiction are not known. In this study, we used rodent models to study behavioral and synaptic correlates related to individual differences in the development of ethanol locomotor sensitization, a form of drug-dependent behavioral plasticity associated with addiction vulnerability. Male Swiss Webster mice were treated daily with saline or 1.8 g/kg ethanol for 21 d. Locomotor activity tests were performed once a week for 15 min immediately after saline or ethanol injections. After at least 11 d of withdrawal, cohorts of saline- or ethanol-treated mice were used to characterize the relationships between locomotor sensitization, ethanol drinking, and glutamatergic synaptic transmission in the nucleus accumbens. Ethanol-treated mice that expressed locomotor sensitization to ethanol drank significantly more ethanol than saline-treated subjects and ethanol-treated animals resilient to this form of behavioral plasticity. Moreover, ethanol-sensitized mice also had reduced accumbal NMDA receptor function and expression, as well as deficits in NMDA receptor-dependent long-term depression in the nucleus accumbens core after a protracted withdrawal. These findings suggest that disruption of accumbal core NMDA receptor-dependent plasticity may represent a synaptic correlate associated with ethanol-induced locomotor sensitization and increased propensity to consume ethanol.

Glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (NMDARs); however, the contributions of NR3A subunit-containing NMDARs to this process have only begun to be considered. Here we characterized the expression of NR3A in the developing mouse forebrain and examined the consequences of NR3A deletion on excitatory synapse maturation. We found that NR3A is expressed in many subcellular compartments, and during early development, NR3A subunits are particularly concentrated in the postsynaptic density (PSD). NR3A levels dramatically decline with age and are no longer enriched at PSDs in juveniles and adults. Genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent with the functional observations, we observed that the deletion of NR3A accelerated the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 in the PSD in postnatal day (P) 8 mice. These data support the idea that glutamate receptors concentrate at synapses earlier in NR3A-knockout (NR3A-KO) mice. The precocious maturation of both AMPAR function and glutamate receptor expression are transient in NR3A-KO mice, as AMPAR currents and glutamate receptor protein levels are similar in NR3A-KO and wildtype mice by P16, an age when endogenous NR3A levels are normally declining. Taken together, our data support a model whereby NR3A negatively regulates the developmental stabilization of glutamate receptors involved in excitatory neurotransmission, synaptogenesis, and spine growth.

Clinical evidence suggests that febrile status epilepticus (SE) in children can lead to acute hippocampal injury and subsequent temporal lobe epilepsy. The contribution of febrile SE to the mechanisms underlying temporal lobe epilepsy are however poorly understood. A rat model of temporal lobe epilepsy following hyperthermic SE was previously established in our laboratory, wherein a focal cortical lesion induced at postnatal day 1 (P1), followed by a hyperthermic SE (more than 30min) at P10, leads to hippocampal atrophy at P22 (dual pathology model) and spontaneous recurrent seizures (SRS) with mild visuospatial memory deficits in adult rats. The goal of this study was to identify the long term electrophysiological, anatomical and molecular changes in this model. Following hyperthermic SE, all cortically lesioned pups developed progressive SRS as adults, characterized by the onset of highly rhythmic activity in the hippocampus. A reduction of hippocampal volume on the side of the lesion preceded the SRS and was associated with a loss of hippocampal neurons, a marked decrease in pyramidal cell spine density, an increase in the hippocampal levels of NMDA receptor NR2A subunit, but no significant change in GABA receptors. These findings suggest that febrile SE in the abnormal brain leads to hippocampal injury that is followed by progressive network reorganization and molecular changes that contribute to the epileptogenesis as well as the observed memory deficits.Copyright © 2011. Published by Elsevier Inc.

Activation of the NMDA glutamate receptors is critical for the initiation of synaptic plasticity. In the developing rat retina, NMDA receptor function has been associated with visual experience, though the light-dependent regulation of the subunit composition of the NMDA receptors is controversial. In the present study, the functional expression of NMDA receptors in the developing rabbit retina was characterized and the impact of light deprivation on how the subunit composition of NMDA receptors is regulated was examined.Antibodies against NR1 and NR2A/B were used to examine neonatal expression patterns of the NMDA receptor subunits. Furthermore, the functional NMDA receptors were mapped using the agmatine (AGB) activation assay.Although NR1 and NR2A/B subunit immunoreactivity was prominently detectable only immediately after birth, AGB activation assay showed that functional NMDA receptors could be identified as early as embryonic day 21. No significant difference was observed between normal- and dark-reared animals in terms of their NR1 and NR2A/B expression. In addition, a comparison of AGB permeation between normal- and dark-reared animals showed no difference in functional expression of NMDA receptors.These results indicate that NMDA receptors participate in the synaptic maturation of retinal circuits during the early stages of development but that the functional NMDA receptors, including their subunit composition, in the developing rabbit retina are independent of the rabbit's visual experience.

Febrile seizures are the most common types of seizure in children, and are generally considered to be benign. However, febrile seizures in children with dysgenesis have been associated with the development of temporal lobe epilepsy. We have previously shown in a rat model of dysgenesis (cortical freeze lesion) and hyperthermia-induced seizures that 86% of these animals developed recurrent seizures in adulthood. The cellular changes underlying the increased risk of epileptogenesis in this model are not known. Using whole cell patch-clamp recordings from CA1 hippocampal pyramidal cells, we found a more pronounced increase in excitability in rats with both hyperthermic seizures and dysgenesis than in rats with hyperthermic seizures alone or dysgenesis alone. The change was found to be secondary to an increase in N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs). Inversely, hyperpolarization-activated cation current was more pronounced in naïve rats with hyperthermic seizures than in rats with dysgenesis and hyperthermic seizures or with dysgenesis alone. The increase in GABAA-mediated inhibition observed was comparable in rats with or without dysgenesis after hyperthermic seizures, whereas no changes were observed in rats with dysgenesis alone. Our work indicates that in this two-hit model, changes in NMDA receptor-mediated EPSCs may facilitate epileptogenesis following febrile seizures. Changes in the hyperpolarization-activated cation currents may represent a protective reaction and act by damping the NMDA receptor-mediated hyperexcitability, rather than converting inhibition into excitation. These findings provide a new hypothesis of cellular changes following hyperthermic seizures in predisposed individuals, and may help in the design of therapeutic strategies to prevent epileptogenesis following prolonged febrile seizures.

S-nitrosylation, as a post-translational protein modification, recently has been paid more and more attention in stroke research. S-nitrosylation regulates protein function by the mechanisms of covalent attachment that control the addition or the removal of nitric oxide (NO) from a cysteine thiol. The derivation of NO is established by the demonstration that, in cerebral neurons, NO mainly generates from neuronal nitric oxide synthase (nNOS) during the early stages of reperfusion. In the past researches, we demonstrate that global ischemia-reperfusion facilitates the activation of glutamate receptor 6 (GluR6) -mediated c-Jun N-terminal kinase (JNK) signaling pathway. The objective of this study is primarily to determine, during the early stages of reperfusion in rat four-vessel occlusion (4-VO) ischemic model, whether nNOS-derived NO affects the GluR6-mediated JNK signaling route via S-nitrosylation which is performed mainly by the biotin switch assay. Here, we show that administration of 7-nitroindazole, an inhibitor of nNOS, or ketamine, an antagonist of N-methyl-d-aspartate receptor (NMDAR), diminishes the increased S-nitrosylation of GluR6 induced by cerebral ischemia-reperfusion. In contrast, 2-amion-5,6-dihydro-6-methyl-4H-1,3-thiazine, an inhibitor of inducible NO synthase does not affect S-nitrosylation of GluR6. Moreover, treatment with sodium nitroprusside (SNP), an exogenous NO donor, increases the S-nitrosylation and phosphorylation of nNOS, leading to the attenuation of the increased S-nitrosylation of GluR6 and the assembling of GluR6* postsynaptic density protein 95 (PSD95)* mixed lineage kinase 3 (MLK3) signaling module induced by cerebral ischemia-reperfusion. The results also show that GluR6 downstream MLK3* mitogen activated protein kinase kinase 4/7* JNK signaling module and nuclear or non-nuclear apoptosis pathways are involved in the above signaling route. However, dithiothreitol (DTT) antagonizes the neuroprotection of SNP. Treatment with DTT alone, as a negative control, prevents S-nitrosylation of proteins, which indicates the existence of endogenously produced S-nitrosylation. These data suggest that GluR6 is S-nitrosylated by endogenous NO in cerebral ischemia-reperfusion, which is possibly correlated with NMDAR* PSD95* nNOS signaling module, and further activates GluR6* PSD95* MLK3 signaling module and JNK signaling pathway. In contrast, exogenous NO donor antagonizes the above action of endogenous NO generated from nNOS. Thus, our results provide the coupling of nNOS with GluR6 by S-nitrosylation during the early stages of ischemia-reperfusion, which can be a new approach for stroke therapy.