Summer Research Fellowship Programme of India's Science Academies

Effect of Mg2+ ions on NMDA receptor mediated translation regulation

Divya Bindra

Department of Biotechnology, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana 500046

Dr. Ravi Muddashetty

Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine (InStem), Bengaluru, Karnataka 560065


Altering the strength of neuronal synapses, termed as synaptic plasticity, forms the mechanistic basis of memory formation and is known to regulate multiple behaviours. At the molecular level, different glutamate receptors are known to mediate various forms of synaptic plasticity over multiple brain regions. Amongst the different glutamatergic receptors, NMDA receptors are known to extensively regulate long-term potentiation (LTP) or long term depression (LTD). De-novo protein synthesis happening in an activity dependent manner is a key requirement for long term forms of synaptic plasticity. Hence, it would be expected that activation of NMDA receptors can generate changes in neuronal protein synthesis to achieve long term effects of plasticity. Inhibition of translation due to NMDAR activation has been previously shown to occur which is largely by the phosphorylation of eukaryotic elongation factor eEF2. NMDA receptor ion channel activity is known to be blocked by extracellular magnesium. We aim to speculate the role of extracellular magnesium in regulating translation inhibition depicted by NMDA receptor activation.

Keywords: synaptic plasticity, Long Term Potentiation (LTP), Long Term Depression (LTD), synaptoneurosome, eEF2


NMDAN-methyl D-aspartate
NMDARN-methyl D-aspartate receptor
AMPAR α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
LTP Long term potentiation 
LTDLong term depression 
mGluRMetabotropic glutamate receptor
iGluRIonotropic glumate receptor
eEF2Eukaryotic elongation factor 2
SNS Synaptoneurosome 
PSD95Post synaptic density 95
GFAPGlial fibrillary acidic protein
MAGUKMembrane associated guanylate kinase



An important property of neuronal function is the activity dependent change in synaptic transmission and modification of the strength of particular synapses, which is known as synaptic plasticity. It is mediated through activation of certain neurotransmitter receptors. This property of synaptic plasticity underpins the molecular mechanisms involved in memory formation and regulation of many behaviours (Takeuchi et al. 2013). Synaptic plasticity has also been attributed to the early formation of neural connections. Additionally, studies of synaptic plasticity impairment have cued its importance in the development of several neuropsychiatric disorders, where defective experience-conditioned rewiring of the brain ensues (Nanou and W. A. Catterall 2018). Thus, diving deep into the mechanistic basis of synaptic plasticity becomes imperative.

The formation of various forms of synaptic plasticity in the context of different glutamate receptors has been widely reported. Glutamatergic receptors are broadly classified as – metabotropic glutamate receptors (mGluRs) and the ionotropic glutamate receptors (iGluRs). Amongst the different ionotropic glutamatergic receptors, NMDA (N-methyl D-aspartate) receptors (NMDARs) have been implicated extensively in regulating long-term potentiation (LTP) or long term depression (LTD) (Hunt and Castillo 2012) (Malenka 2012). Another key finding shedding light over the mechanisms regulating synaptic plasticity was the requirement of novel protein synthesis to mediate long term potentiation (LTP) in hippocampus (Krug et al., 1984). Studies in rodents, similarly, reported the requirement of de-novo transcription as well as translation for long term memory consolidation (McGaugh, 2000; Kandel, 2001). In vitro studies have also shown the precondition of protein synthesis for mediating long term forms of plasticity (Sutton and Schuman, 2006; Rangaraju et al. 2017). These observations have been further supported by studies wherein site directed genetic disruption of different translational signalling molecules impair the formation of long-term plasticity and memory (Costa-Mattioli et al., 2009). Hence, from these observations it would be expected that activation of NMDA receptors can generate enduring changes in neuronal protein synthesis to alter synaptic strength over longer period of time.

NMDARs are multimeric protein complexes composed of different subunits. This cation channel complex uniquely comprises of various receptor and modulatory domains which include the ligand L-glutamate binding site and a glycine binding site (Traynelis et al. 2010). These receptors are permeable to calcium ions, along with sodium and barium ions. These ligand-gated ion channels are blocked by a channel resident Mg2+ ion in voltage dependent manner. Its activation requires presence of extracellular glutamate and depolarization of membrane potential simultaneously. At the basal membrane potential, the magnesium ions occupy the channel pore and block the glutamate mediated ionic entry through NMDA receptors (Traynelis et al. 2010). When depolarised (mostly through AMPAR receptors), the magnesium ions are displaced and the block is surpassed to allow influx of calcium ions for mediating further downstream functions. Thus, it can act as a co-incidence detector of both pre and post synaptic activity.

    Model of synaptic transmission at excitatory synapses: During basal synaptic transmission (left panel), synaptically released glutamate binds both the NMDA and AMPARs. Na+ flows through the AMPAR channel but not through the NMDAR channel because of the Mg2+ block of this channel. Depolarization of the postsynaptic cell (right) relieves the Mg2+ block of the NMDAR channel and allows both Na+ and Ca2+ to flow into the dendritic spine. The resultant increase in Ca2+ in the dendritic spine is necessary for triggering the subsequent events that drive synaptic plasticity.

    Statement of the Problems

    While channel activity has been shown to be a function of presence or absence Mg2+, recent research has indicated that NMDAR mediated synaptic signalling can be a result of alternative signalling mechanisms independent of channel function (Weilinger et al. 2016; Dore et al. 2016). NMDAR induced LTD has been observed in the absence of ion flow (Nabavi et al. 2013; Stein et al. 2015). Furthermore, amyloid-beta induced synaptic depression has been shown to be NMDAR activity dependent exclusive of ion flow (Kessels et al. 2013; Birnbaum et al. 2015). However, it is not well understood whether the Mg2+ blockage of NMDARs has any effect over signalling mechanisms regulating translation.

    Objectives of the Research

    This research aimed to compare the effects of NMDAR stimulation by NMDA (specific agonist) in the presence or absence of extracellular Mg2+. Previous experiments in the lab have established that NMDAR stimulation leads to an immediate (~2 mins) inhibition of global translation achieved mostly by enhanced phosphorylation of eEF2. Thus, the phospho to total eEF2 ratio increases significantly on NMDAR stimulation. Therefore, we used phosphorylation of eEF2 as proxy to assess the state of global translation and to speculate the impact of Mg2+ in regulation of NMDAR dependent translation.


    A review by Citri and Malenka discusses about various forms of synaptic plasticity including different types of short term and long-term plasticity. They interestingly describe the basic properties of cooperativity, associativity and input specificity illustrated by LTP, relating it to be a possible mechanistic basis of memory storage. They report NMDAR dependent LTP and LTD being majorly studied depicting the bidirectional synaptic control exerted by NMDA receptors. They also acknowledge the alteration of proteome at synapses for maintenance of these long term type of plasticity (Citri and Malenka 2008). Another review by Ho et al talks about hippocampal synaptic plasticity and the ability of different stimuli to evoke varying changes in synaptic strength, high frequency stimuli being responsible for eliciting LTP while low frequency stimuli elicit LTD. They also talk about the temporal functioning of plasticity and the requirement of protein synthesis by long term forms of plasticity (Ho et al. 2012). These observations enforce the importance of NMDAR dependent regulation of translation for modulating synaptic strength.

      A- Hippocampus region in a rodent brain; B- Different regions in the hippocampus (transverse section); C,D- Trains of high frequency stimulation (HFS) or low frequency stimulation (LFS) to the axonal fibers produce sustained increases or decreases, respectively of the EPSP amplitude in response to subsequent test stimuli. These forms of plasticity are known as long-term potentiation (LTP) and long-term depression (LTD).

      Furthermore, an experiment on rodent synaptic preparations by Scheetz et al has shown the effect of NMDAR activation being responsible for a decrease in total protein synthesis mediated by a subsequent increase in eEF2 phosphorylation (Scheetz et al. 2000). Eukaryotic elongation factor 2 (eEF2) is a salient GTP binding molecule responsible for peptidyl-tRNA translocation from ribosomal A site to P site during protein chain elongation (Moldave 1985). Studies on chemically induced LTP have also similarly observed its regulation at the translation elongation step (Chotiner et al. 2003). These observations alongwith previous findings in the lab emphasise on eEF2 factor being an important regulator and indicator of the overall translational state of the cell.

      A review by Hansen et al discusses about the different subunit composition of NMDA tetrameric receptors and their modulation. They describe the NMDA channel pore structure and the possible structural influences of the Mg2+ block which has been potentially shown to be dependent on S/L site in M3 transmembrane region of GluN2 subunits, although clear structural insights are absent (Hansen et al. 2018).

        General pore structure of NMDA receptors

        The ionotropic properties of NMDA receptors have long been established through their ion channel mediated functions but a few recent reports have also ascertained the ability of NMDARs to function in the absence of ion-flux. These reports suggest the possible metabotropic functioning of NMDA receptors thereby solubilizing the line of demarcation between the two classes of glutamate receptors. One such study by Nabavi et al describes the production of LTD after pharmacological blockage of ionic movement through NMDAR (Nabavi et al. 2013). This study reports LTD being observed after the block of ion channel by MK801 and 7-CK (act as antagonists for glycine binding GluN1 subunits) but absence of LTD when the ion channel is blocked using AP-5 (antagonist for glutamate binding GluN2 subunit). These results depict the importance of ligand binding but not channel activity to induce one form of NMDAR mediated synaptic plasticity.

          NMDAR metabotropic function model

          These observations regarding probable difference in NMDAR functioning mechanisms impels to question whether the magnesium block of NMDAR ion channel plays any role on the observed downstream effect of NMDA functioning that leads to decreased global protein synthesis (as depicted by increased phosphorylation of eEF2), which this research addresses.



          Antibodies and drugs used

          Antibody/drug Dilution
          Anti-phospho eEF2 antibody (Thr 56) 1:1000 dilution for western blotting analysis
          Anti-eEF2 antibody 1:1000 dilution for western blotting analysis
          Anti-tubulin antibody 1:3000 dilution for western blot analysis
          Anti-synapsin 1 antibody 1:1000 dilution for western blotting analysis
          Anti-PSD95 antibody 1:1000 dilution for western blotting analysis
          Anti-GFAP antibody 1:1000 dilution for western blotting analysis
          Anti-rabbit HRP labelled secondary antibody 1:5000 dilution for western blotting analysis
          Anti-mouse HRP labelled secondary antibody 1:5000 dilution for western blotting analysis
          NMDA 40μM for SNS stimulation


          Synaptoneurosome preparation

          For this experiment, synaptoneurosomes prepared from P30 (postnatal day 30) Sprague Dawley WT rat cortical neurons were used.

          Rat cortices were first dissected in pre-chilled [1X] PBS followed by homogenisation of the cortical tissue at 4°C in 7 volumes of synaptoneurosome(SNS) buffer.

          • SNS Buffer composition:
          Components Working Concentration (mM)
          Tris-HCl pH 7.4 25
          NaCl 118
          KCl 4.7
          MgSO4 1.2
          CaCl2 2.5
          KH2PO4 1.53
          Glucose 212.7
          Complete protease inhibitors (Roche)

          150µl of this homogenate was collected separately for SNS prep validation through western blot analysis, while the rest of the homogenate was passed through three 100μm nylon mesh filters and one 10μm filter kept in a single filter holder. Two equal fractions were collected per animal following centrifugation at 1000g for 20 minutes at 4°C. The pellet in one fraction was re-dissolved in 1.5ml of SNS buffer (with Mg2+) and the other in 1.5ml of SNS buffer (without Mg2+). 150µl of this SNS prep was collected separately of SNS validation.

           Synaptoneurosome stimulation

          Stimulation was done after prewarming the SNS samples at 37°C for 10 minutes. For this, 250µl fractions were collected in two separate tubes for samples containing Mg2+. Likewise was done for samples without Mg2+. The first fraction was taken out and kept on ice for a few seconds followed by a short spin for 25-30 seconds. The supernatant was discarded and pellet was resuspended in 400µl lysis buffer. This aliquot was considered for basal/0 min reading. Then, stimulation was done for the other fraction using 40μM NMDA at 37°C for 5 minutes. The stimulus was removed by incubation in ice for a few seconds followed by short spin of samples for 25-30 seconds and addition of 400µl of lysis buffer to the pellet after 5 minutes (5’ readout). These samples were then processed further for western blot analysis.


          Precast gel with a concentration gradient of 4-12% was used for SDS-PAGE. The reagents were prepared and samples were loaded after adding Laemmli sample buffer followed by heating of samples at 95°C for 5 minutes. The gel was run in the gel running buffer at a constant voltage of 75V till the tracking dye reached the gel base.

          • SDS-PAGE buffer composition
          • Laemmli sample buffer (4X)
          Components Concentration
          10% SDS 8%
          Glycerol 40%
          1M Tris-Cl (pH6.8) 250mM
          Bromophenol blue 0.02%
          β-mercaptoethanol 8%

          2. Running buffer (10X)

          Components Concentration
          Tris-HCl (pH8.3) 25mM
          Glycine 200mM
          SDS 0.1%

          The running buffer was diluted to [1X] concentration before use.

          Western blot

          SDS-PAGE was followed by western transfer on PVDF membrane pre-activated using methanol. The western transfer was performed for 3 hours at a constant current of 380mA at 4°C and further western blotting was carried out using the above mentioned antibodies to bind to respective protein bands. Blocking of the membrane was done using 5% freshly prepared BSA solution.

          Protein Ladder used: Precision plus protein dual color standard (10-250KDa)


          1.   Transfer buffer(10x)

          Component Amount
          Tris base 22.5g
          Glycine 108g
          Volume adjusted to 1L using milliQ water

           The transfer buffer was diluted to [1X] concentration before use.

          2.   TBS pH 7.6 (10X)

          Component Amount
          Tris base 24g
          NaCl 88g
          Dissolved the components in ~900ml water and pH adjusted to 7.6. Final volume was made to 1L.

           To 100ml of 10X TBS, 1ml of Tween-20 (0.1%) was added and volume made upto 1L to make [1X] TBST washing buffer for use.

          Blot analysis

          Fiji (Image J based image processing package) was used for image analysis obtained after western blot development.

          Statistical analysis

          Statistical analysis and data plotting were done using GraphPad Prism (Prism 7.01, Graphpad Software Inc., La Jolla, CA, USA).


          Validation of Synaptoneurosome Preparation

          Synaptoneurosome preparation from P30 rat cortices were validated by determining the enrichment of synaptic markers PSD 95 and Synapsin 1 and glial marker GFAP in the SNS preparation when compared with total cortical lysate.

          For this, the samples separately collected during homogenate and SNS preparation were processed for SDS-PAGE followed by Western blot analysis and their presence was detected by labelling with HRP labelled secondary antibodies for anti-synapsin 1, anti-GFAP and anti-PSD95 antibodies. The blots were then developed using luminol and hydrogen peroxide as substrate solution. These were then quantified to detect for their enrichment in SNS preparations.

          PSD95(post synaptic density 95) is a 95kDa protein, member of MAGUK family of proteins containing PDZ domain. Its structure comprises three PDZ domains, an SH3 domain, and a guanylate kinase-like domain (GK) with interspersed linker regions. It is almost exclusively present post-synaptically (Hunt et al,1996) and is important for anchoring of other proteins. Thus, it represents an excellent post-synaptic marker. Synapsin 1 is a 74kDa protein which forms the major peripheral protein component of the synaptic vesicles at nerve endings (Gerald Thiel 1993) and is involved in modulation of neurotransmitter release. Thus, it forms an excellent pre-synaptic marker. GFAP is an intermediate filament protein (50kDa) present in many types of glial cells and therefore represents a glial marker.

            Immunoblot showing changes in the levels of synaptic markers (PSD95, Synapsin 1) along with glial marker (GFAP) in cortical total lysate and synaptoneurosome preparation. In each case, the levels were normalised to tubulin before comparison.
            Relative levels (fold change) of the synaptic markers when compared with total lysate
            Synapsin 1 PSD95 GFAP
            Cortex total lysate 1 1 1
            Cortical Synaptoneurosomes 1.619736 1.730202 0.996236
              Bar graph showing normalised levels of Synapsin1, PSD95 and GFAP in total cortical lysate and SNS

              ​We observed a ~73% enrichment of post-synaptic marker PSD 95 and ~62% enrichment of pre-synaptic marker Synapsin 1. No enrichment could be observed for glial marker GFAP. This shows the major components of this biochemical preparation. The preparation is not the purest (Muddashetty et. al., 2007) as there are other purer preparation protocols available based on various density gradient centrifugations. The advantage of this crude preparation is that it can be made faster than the other protocols and can therefore be stimulated to investigate activity mediated changes in the synaptic compartments.

              NMDA Stimulation and the Effect of Mg2+ on Cortical Synaptoneurosomes

              Cortical synaptoneurosomes were pre-incubated at 37°C for 5 minutes in presence or absence of extracellular Mg2+ (1.2mM). They were then stimulated with NMDA (40μM) for 5 minutes. This was followed by immediate removal of the NMDA containing media and lysis of synaptoneurosomes with lysis buffer. The lysates were then used for western blot analysis for quantification of phospho to total eEF2 levels. The samples were run in duplicates for probing of phospho eEF2 and total eEF2, each of them individually normalized to their respective tubulin levels which acted as loading control. These normalized values were then used to calculate the phospho to total eEF2 ratio. The change in the ratio was measured by comparing the NMDA stimulated samples with their respective unstimulated control samples.

                Immunoblot showing changes in levels of phospho and total eEF2 in unstimulated (representing basal values) and NMDA stimulated (for 5 minutes) samples with or without extracellular magnesium. Both the levels were normalised to tubulin before calculating the ratio. 

                When compared, phospho to total eEF2 ratio was found to be increased on NMDAR stimulation compared to unstimulated control SNS prep both in presence and absence of Mg2+ (Graph II). This helps us to infer that the activity mediated response on eEF2 phosphorylation stays unperturbed in absence of Mg2+. Therefore, absence of Mg2+ did not abrogate NMDA mediated translation inhibition. Interestingly, we found an increase in basal eEF2 phosphorylation in absence of Mg2+ (Graph III). This is probably due to increased opening of NMDA receptors basally in absence of Mg2+. Therefore, our experiments indicate two important aspects of translation regulation. In unstimulated conditions, basal translation depends on the fraction of NMDAR population in open configuration and therefore dependent on various parameters regulating NMDAR channel opening.

                While the stimulation dependent changes in global translation could be independent of whether of the NMDAR channel states. An alternate explanation could be that NMDA binding to the receptor can lead to a subsequent removal in absence of any co-agonist or co-incident depolarizations.

                  Bar graph representing fold change of phospho/total eEF2 ratio in NMDA stimulated samples in presence or absence of magnesium.
                    Bar graph representing changes in phospho/total eEF2 levels of unstimulated and NMDA stimulated samples in presence or absence of magnesium. All samples are normalised to the basal levels in presence of magnesium.


                    SNS Preparation was Successfully Validated

                    From the observed increase in post and pre-synaptic markers (PSD95 and Synapsin 1, respectively) in the SNS preparation without any increase in the glial marker i.e. GFAP when compared with their levels in the total cortical lysate, it can be successfully validated that the SNS preparations were fairly enriched in the synaptic compartments to carry out further experimentation to be observed at neural synapses.

                    Influence of Extracellular Magnesium on eEF2 Phosphorylation Levels and NMDA Receptor Mediated Translation Inhibition

                    In the present study we have demonstrated that the absence of extracellular magnesium causes a basally increased NMDAR mediated inhibition of protein synthesis in synaptoneurosomes from rat cortices. It was also observed that NMDA stimulation further enhances translation inhibition in presence or absence of magnesium, as was observed by an increase in the level of phosphorylated eEF2. Increased eEF2 phosphorylation response is known to reduce the rate of elongation during translation thereby causing depressed protein synthesis.

                    Since extracellular magnesium ions are known to normally exert a block of NMDAR ion channel activity according to previous studies, an absence of extracellular Mg2+ may result in an increased probability of opening of NMDARs basally. Thus an increased fraction of NMDA receptors in open channel state take part in regulation of global translation at the basal level. This can be a plausible explanation due to which basal elevation of phosphorylated eEF2 was observed.

                    Upon stimulation by NMDA as a specific agonist for NMDA receptors, further elevation in phospho to total eEF2 was observed independent of whether extracellular magnesium was present or absent. This depicts that the NMDAR mediated inhibition of protein synthesis is not abolished by absence of extracellular Mg2+. One possible explanation for the observed increase in NMDAR mediated translation inhibition could be that the agonist binding results in a subsequent removal of magnesium in absence of any co-agonist or co-incident depolarization but the exact molecular mechanistic basis for NMDA induced NMDAR activation still remains to be explored. Alternatively, some recent studies have reported the ion channel independent activity of NMDARs, indicating a probable functioning of alternative signalling mechanisms to exert NMDA receptor downstream effects including some forms of NMDAR dependent synaptic plasticity. Our results showing NMDAR mediated global translation inhibition independent of extracellular magnesium ions may further support these findings. An increased eEF2 phosphorylation by NMDAR activation independent of its ion channel opening may be a probable explanation. Therefore, our results strongly hint towards the fact that NMDAR dependent translation inhibition is independent of extracellular Mg2+.


                    It can be concluded from the work that in cortical synaptoneurosomes, the global translation inhibition mediated by NMDA receptors is independent of extracellular magnesium ions. It can also be ascertained that the presence of extracellular magnesium ions determines NMDAR modulated basal levels of global protein synthesis.

                    However, further studies are needed to be carried out to corroborate the findings of the present study. Studies measuring the alteration in downstream effects of NMDAR activation on translation at varied time points after stimulation at synapses may be performed. Also, the influence of extracellular magnesium concentrations on activity dependent effects of NMDAR stimulation on global protein synthesis can be observed in cultured cortical neurons for greater physiological and mechanistic insights.


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                    I would like to thank the Indian Academy of Sciences (IASc – INSA) for providing me the opportunity to work under the Summer Research fellowship programme in the Institute for Stem Cell Biology and Regenerative Medicine (InStem) and for providing me a place of stay to carry out my project work conveniently.

                    I extend my heartfelt gratitude to my guide, Dr. Ravi Muddashetty, for providing me the opportunity to work in his lab under his excellent supervision. I’m deeply indebted to him for his vital support and valuable advice and motivation throughout this project. I would like to express my sincere thanks to my mentor, Sudhriti Ghosh Dastidar for guiding me with his knowledge. I am very grateful to him for his boundless efforts and supervision and for the critical assessment of my project report. I am also grateful to all other lab members for their encouragement and constant support.

                    Finally, I express my deep gratitude and appreciation to my parents for their incessant love and support.


                    • Fig 1: Synaptic Plasticity: Multiple Forms, Functions, and Mechanisms: A Citri and RC Malenka, 2008 
                    • Fig 2: The Cell Biology of Synaptic Plasticity; Ho, Lee and Martin 2012
                    • Fig 3: Structure, Function, and Allosteric Modulation of NMDA Receptors: Hansen et al. 2018
                    • Fig 4: The Emergence of NMDA Receptor Metabotropic Function: Insights from Imaging; Dore, Aow and Malinow 2016 
                    Written, reviewed, revised, proofed and published with