Background & Aims

Metabotropic glutamate receptor 5 (mGluR5) is a G protein-coupled receptor (GPCR) expressed in the pain neuroaxis 1. In animal models, mGluR5 agonists are pro-nociceptive, while antagonists are anti-nociceptive (2,3). In spinal cord dorsal horn (SCDH) neurons, mGluR5 is present at both the cell surface and on intracellular membranes (4). Intracellular mGluR5 exhibits unique and nociception-relevant downstream signalling compared to cell surface mGluR5 (5-7). Previous pharmacological studies have implicated intracellular mGluR5 in persistent pain as cell permeable but not impermeable antagonists effectively reduce neuropathic and inflammatory injury-induced pain hypersensitivity, as well as pain-induced enhancements of SCDH signalling (4,8). To overcome limitations of pharmacological manipulation we aim to determine whether genetic restriction of mGluR5 to intracellular membranes (mGluR5IM mice) produces changes in measures of pain behaviour and downstream nociceptive signalling.

Methods

mGluR5IM mice were generated by inserting an endoplasmic reticulum/ nuclear membrane targeting motif from the Lamin-B receptor upstream of the mGluR5 C-terminal stop codon using CRISPR. Loss of cell surface mGluR5 was validated anatomically and functionally. Here, mGluR5IM and WT mice of both sexes were injected with Complete Freund’s Adjuvant (CFA) or saline control, to the left hind paw. At 3 days post-injection mice were injected intrathecally with 30 µg glutamate and video recorded for 60 minutes. Videos were analysed for glutamate-induced nociceptive behaviours by measuring the time spent licking or biting the hind body, as well as assessing mouse grimace scale scores. Two hours post-injection spinal cords were removed, and L3-5 sections were prepared for immunohistochemistry of targets downstream of intracellular mGluR5: pERK1/2, Arc/Arg3.1 and c-fos. The number of puncta of each target was quantified with ImageJ. All data were compared across genotype, sex and injury conditions.

Results

CFA injected mice show increased glutamate-induced nociceptive behaviour when compared to saline mice, in agreement with previous rat studies. Interestingly, mGluR5IM mice demonstrate increased glutamate-induced nociceptive behaviours in comparison to WT mice. This is observed in both parameters measured with an increase in the time spent licking/ biting the hind body and an increase in mean mouse grimace scale scores in mGluR5IM mice compared to WTs. Preliminary data suggests that this behavioural finding corroborates with alterations in signalling downstream of intracellular mGluR5 in the SCDH, with increased pERK1/2, Arc/Arg3.1 and c-fos staining in mGluR5IM mice, compared to WTs, following intrathecal glutamate injection.

Conclusions

These findings support the importance of the intracellular localisation of mGluR5 in pain modulation. Despite the GPCR field’s canonical view of the primacy of cell surface receptors, this work highlights that GPCRs located on intracellular membranes are functional and can have significant impacts on nociceptive signalling and behaviour. Future work will further assess the downstream signalling of intracellular mGluR5 following intrathecal glutamate injection using a combination of immunohistochemistry and western blot in both inflammatory and neuropathic models of pain in mGluR5IM mice.

References

1.Alvarez, F.J., Villalba, R.M., Carr, P.A., Grandes, P. and Somohano, P.M., 2000. Differential distribution of metabotropic glutamate receptors 1a, 1b, and 5 in the rat spinal cord. Journal of Comparative Neurology, 422(3), pp.464-487.
2.Fisher, K., Lefebvre, C. and Coderre, T.J., 2002. Antinociceptive effects following intrathecal pretreatment with selective metabotropic glutamate receptor compounds in a rat model of neuropathic pain. Pharmacology Biochemistry and Behavior, 73(2), pp.411-418.
3.Pitcher, M.H., Ribeiro?Da?Silva, A. and Coderre, T.J., 2007. Effects of inflammation on the ultrastructural localization of spinal cord dorsal horn group I metabotropic glutamate receptors. Journal of Comparative Neurology, 505(4), pp.412-423.
4.Vincent, K., Cornea, V.M., Jong, Y.J.I., Laferrière, A., Kumar, N., Mickeviciute, A., Fung, J.S., Bandegi, P., Ribeiro-da-Silva, A., O’Malley, K.L. and Coderre, T.J., 2016. Intracellular mGluR5 plays a critical role in neuropathic pain. Nature communications, 7(1), p.1-13.
5.Jong, Y.J.I., Kumar, V. and O’Malley, K.L., 2009. Intracellular metabotropic glutamate receptor 5 (mGluR5) activates signaling cascades distinct from cell surface counterparts. Journal of Biological Chemistry, 284(51), pp.35827-35838.
6.O’Malley, K.L., Jong, Y.J.I., Gonchar, Y., Burkhalter, A. and Romano, C., 2003. Activation of metabotropic glutamate receptor mGlu5 on nuclear membranes mediates intranuclear Ca2+ changes in heterologous cell types and neurons. Journal of Biological Chemistry, 278(30), pp.28210-28219.
7.Kumar, V., Jong, Y.J.I. and O’Malley, K.L., 2008. Activated nuclear metabotropic glutamate receptor mGlu5 couples to nuclear Gq/11 proteins to generate inositol 1, 4, 5-trisphosphate-mediated nuclear Ca2+ release. Journal of Biological Chemistry, 283(20), pp.14072-14083.
8.Vincent, K., Wang, S.F., Laferrière, A., Kumar, N. and Coderre, T.J., 2017. Spinal intracellular metabotropic glutamate receptor 5 (mGluR5) contributes to pain and c-fos expression in a rat model of inflammatory pain. Pain, 158(4), pp.705-716.
9.Lynch, M.E., 2011. The need for a Canadian pain strategy. Pain Research and Management, 16, pp.77-80.
10.Dölen, G. and Bear, M.F., 2008. Role for metabotropic glutamate receptor 5 (mGluR5) in the pathogenesis of fragile X syndrome. The Journal of physiology, 586(6), pp.1503-1508.
11.Matosin, N. and Newell, K.A., 2013. Metabotropic glutamate receptor 5 in the pathology and treatment of schizophrenia. Neuroscience & Biobehavioral Reviews, 37(3), pp.256-268.

Presenting Author

Roseanna Rought

Poster Authors

Roseanna Rought

MSc

McGill Univeristy

Lead Author

E-mail

Cici Chen

McGill University

Lead Author

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Delara Moussavi

McGill University

Lead Author

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Maya Jammoul

McGill University

Lead Author

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Manon St-Louis

McGill University

Lead Author

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Karen L. O'Malley

PhD

Washington University School of Medicine

Lead Author

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Alfredo Ribeiro da Silva

McGill University

Lead Author

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Terence J. Coderre

PhD

McGill University

Lead Author

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Topics

  • Models: Chronic Pain - Inflammatory