A series of studies has shown the importance of AMPA-type glutamate receptors (AMPARs) for memory formation. The aim of the current study was to show whether GluR1 and GluR2 complexes rather than subunits in mouse hippocampus were involved in training in the multiple T-Maze (MTM). C57BL/6J mice were trained in the MTM and compared to yoked controls. 6 h following the completion of the fourth day training, mice were euthanized, hippocampi were taken and proteins extracted, run on blue native gels with subsequent immunoblotting with antibodies against mouse GluR1 and GluR2. On blue-native western blotting, GluR1 protein was represented by a single band at the apparent molecular weight of about 480 kDa probably indicating a tetrameric assembly. GluR2 protein was represented by a single band between apparent molecular weights of 480 and 720 kDa indicating a homo- or heteropolymer probably with other AMPAR or regulatory subunits. In mice trained in the MTM, protein levels for GluR1 were significantly increased while GluR2 levels were significantly decreased. On two-dimensional (2D) gel electrophoresis, the presence of GluR1 and GluR2 were identified by mass spectrometry, and 2D immunoblotting revealed several expression forms of these receptor subunits. Findings unequivocally show that GluR1 and GluR2 complexes are linked to training in the MTM in C57BL/6J mice. These results may not only form the basis for studying receptor complexes rather than receptor subunits in memory formation or mechanisms of potential cognitive enhancers but represent a tool for investigations into pharmacological studies including the use of glutamate receptor agonists and antagonists.
There is limited information on the role of GABA type A receptors (GABARs) containing α1, α5 and γ2 subunits in learning and memory. Here, we assessed the possible role of such receptors in spatial learning using the multiple T-maze (MTM) paradigm. C57BL/6J mice were trained in the MTM which induced elevated levels of α1 and α5 subunit-containing hippocampal GABAR complexes. Moreover, spatial learning evoked a significant increase in the colocalization of α1 and α5 subunits in both, CA1 and dentate gyrus regions of the hippocampus suggesting the formation of complexes containing both subunits. Additionally, the presence of α1, α5 and γ2 subunits in high molecular weight GABARs was detected and significant correlation in the level of α1-containing complexes with those containing α5 and γ2 subunits was demonstrated. Accordingly, α1 deficiency led to decreased levels of γ2 subunit-containing complexes, however, had no effect on α5-containing ones. On the other hand, α1 knockout mice showed impaired performance in the MTM correlating with increased levels of α5 subunit-containing GABARs in comparison to trained floxed control animals which quickly learned the task. Taken together, these results suggest that α1, α5 and γ2-containing hippocampal GABAR complexes play an essential role in spatial learning and memory in which targeted disruption of the α1 subunit produces profound deficits.
The NMDA receptor (NMDA-R) is a key element in neural transmission and mediating a vast variety of physiological and pathological processes in the nervous system. It is well-known that phosphorylation is required for functioning of the NMDA-R, and we therefore decided to study this post-translational modification in subunits NR1 and NR2A-D. Immunoprecipitation with an antibody against NR1 was carried out from rat hippocampi and SDS-PAGEs were run. Bands were punched, destained, and digested with trypsin and chymotrypsin and peptides were identified by nano-LC-ESI-MS/MS using an ion trap (HCT). Proteins were identified using specific software. Phosphorylations were verified by phosphatase treatment and reanalysis by mass spectrometry. The NMDA-R subunits NR1 and 2A-D were identified. On NR2A, a novel phosphorylation site was observed at S511, and on NR2B, four novel phosphorylation sites were revealed at S886, S917, S1303, and S1323 by mass spectrometry and verified by phosphatase treatment with mass spectrometrical reanalysis. A series of NMDA-R phosphorylations have been reported and these serve different functions as receptor activation, localization, and protein-protein interactions. Herein, findings of novel phosphorylation sites are extending knowledge on chemical characterization of the NMDA-R and warrant studying function of site-specific receptor phosphorylation in health and disease.
Reduced daily intake of magnesium (Mg(2+)) is suggested to contribute to depression. Indeed, preclinical studies show dietary magnesium restriction (MgR) elicits enhanced depression-like behaviour establishing a causal relationship. Amongst other mechanisms, Mg(2+) gates the activity of N-methyl-D-asparte (NMDA) receptors; however, it is not known whether reduced dietary Mg(2+) intake can indeed affect brain NMDA receptor complexes. Thus, the aim of the current study was to reveal whether MgR induces changes in brain NMDA receptor subunit composition that would indicate altered NMDA receptor regulation. The results revealed that enhanced depression-like behaviour elicited by MgR was associated with reduced amygdala-hypothalamic protein levels of GluN1-containing NMDA complexes. No change in GluN1 mRNA levels was observed indicating posttranslational changes were induced by dietary Mg(2+) restriction. To reveal possible protein interaction partners, GluN1 immunoprecipitation and proximity ligation assays were carried out revealing the expected GluN1 subunit association with GluN2A, GluN2B, but also novel interactions with GluA1, GluA2 in addition to known downstream signalling proteins. Chronic paroxetine treatment in MgR mice normalized enhanced depression-like behaviour, but did not alter protein levels of GluN1-containing NMDA receptors, indicating targets downstream of the NMDA receptor. Collectively, present data demonstrate that dietary MgR alters brain levels of GluN1-containing NMDA receptor complexes, containing GluN2A, GluN2B, AMPA receptors GluA1, GluA2 and several protein kinases. These data indicate that the modulation of dietary Mg(2+) intake may alter the function and signalling of this receptor complex indicating its involvement in the enhanced depression-like behaviour elicited by MgR.
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