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Dietary magnesium restriction reduces amygdala–hypothalamic GluN1 receptor complex levels in mice

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An Erratum to this article was published on 26 June 2014

Abstract

Reduced daily intake of magnesium (Mg2+) 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, Mg2+ gates the activity of N-methyl-d-asparte (NMDA) receptors; however, it is not known whether reduced dietary Mg2+ 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 Mg2+ 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 Mg2+ 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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Acknowledgments

This work was funded partially by the Austrian Science Fund (FWF): P22931-B18 (to NS and GL), W 1206-B18 SPIN (to NS) and partially sponsored by the Verein zur Durchführung der wissenschaftlichen Forschung auf dem Gebiet der Neonatologie und Kinder intensiv Medizin.

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Correspondence to Gert Lubec.

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M. Ghafari, N. Whittle contributed equally to this work.

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Supplemental Fig. 1. A single band was representing the GluN2A-containing receptor complex; levels were comparable between groups. Bars in supplemental Figs. 1, 3 represent standard deviations. Representative images of Western blots 1a,b controls, 2a,b MgR, 3a,b MgR + paroxetine treatment.

Supplemental Fig. 2. A single band was representing the 5-HT1A and levels were comparable between groups.

Supplemental Fig. 3. (A) Behavioral outcome from animals used for the mRNA GluN1 steady state levels. (B) The results from the real-time PCR experiments are demonstrating comparable mRNA-GluN1-steady state levels.

Supplemental Fig. 4. Detection of GluN1-GluA1 and GluN1-GluA2 receptor complexes by “In Situ” Proximity Ligation Assay (PLA).

Representative images showing the GluN1-GluA1(A) and GluN1-GluA2 (B) receptor complex with DAPI counterstained nuclei in mouse hypothalamus. Each PLA signal represents the interaction of a single GluN1 receptor with a GluA1, GluA2 receptor respectively. The arrows indicate examples of PLA signals. Confocal microscope images acquired at 20X magnification. Scale bar 10 µm.

Supplemental Table 1. shows the significant correlation characteristics between receptor complexes.

Supplemental Table 2. reveals that no significant correlation between receptor complexes and parameters from the FST were obtained.

Supplemental Table 3. Peptide sequences that were used for the mass spectrometrical identification of GluN1-receptor complex constituents are given.

Supplementary material 1 (JPEG 72 kb)

Supplementary material 2 (JPEG 26158 kb)

Supplementary material 3 (JPEG 77 kb)

Supplementary material 4 (JPEG 59 kb)

Supplementary material 5 (PPT 68 kb)

Supplementary material 6 (PPT 109 kb)

Supplementary material 7 (XLS 99 kb)

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Ghafari, M., Whittle, N., Miklósi, A.G. et al. Dietary magnesium restriction reduces amygdala–hypothalamic GluN1 receptor complex levels in mice. Brain Struct Funct 220, 2209–2221 (2015). https://doi.org/10.1007/s00429-014-0779-8

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