Background & Aims

Neuropathic pain (NP) is a chronic condition resulting from damage to the somatosensory nervous system1. We investigate how spinal dorsal horn (DH) neuronal pathways involved in the coding of sensory information can be targeted to relieve pain. While evidence suggests that deficits in DH inhibitory transmission (disinhibition) result in symptoms of NP2, directly targeting this disinhibition remains challenging. In recent studies, we discovered that, following nerve injury, microglia mediate the elimination (pruning) of DH synapses, resulting in a net loss of inhibitory synapses3. While the mechanism that dictates the selectivity of inhibitory synapse removal in NP remains unknown, a study showed during postnatal development, brain microglia expressing GABA B receptors (GABABRs) were responsible for sculpting inhibitory synapses4. We thus, aim to investigate this process in a mice model of NP by quantifying GABABRs on DH microglia and assessing synaptic engulfment by these microglia.

Methods

Adult TMEM119-tdTomato mice5 having either spared nerve injury, modelling peripheral neuropathy6, or sham surgery are used to facilitate the visualization of microglia in DH laminae I and II, at several time points. Engulfment of excitatory (as defined by presence of VGLUT2) and inhibitory (VGAT+) synapses7 by microglia expressing GABABRs are assessed using high resolution confocal microscopy. The volume of synaptic markers within the microglial lysosomes (labelled with CD68) are quantified using the IMARISs software. Antibodies against GABABR1and GABABR2 are used to identify microglia colocalizing these receptors.

Results

Our initial data shows that GABABR1 is upregulated on microglial membranes in Lamina II ipsilateral to the injury side a week post-SNI. This suggests that both GABABRs might be upregulated and colocalized in the microglia on the injured side at different time points after surgery. Future steps include showing the relative engulfment of excitatory and inhibitory synapses by these GABABRs-positive microglia and seeing how genetically ablating this subpopulation of microglia in the spinal cord will affect the NP phenotype.

Conclusions

These experiments help identify the specific elements of the DH circuitry undergoing inhibitory synaptic pruning and help narrow down the search for the mechanistic underpinning of the maladaptive response to nerve injury. Furthermore, knowing the role that the GABABRs-expressing microglia play in this DH plasticity may help in the design of future therapeutic approaches.

References

1. Cavalli, E., Mammana, S., Nicoletti, F., Bramanti, P., & Mazzon, E. The neuropathic pain: An overview of the current treatment and future therapeutic approaches. Int J Immunopathol Pharmacol 33, 2058738419838383, (2019).
2. Price, T. J. & Prescott, S. A. Inhibitory regulation of the pain gate and how its failure causes pathological pain. Pain 156, 789-792, (2015).
3. Yousefpour, N., Locke, S., Deamond, H., Wang, C., Marques, L., St-Louis, M., Ouellette, J., Khoutorsky, A., De Koninck, Y., & Ribeiro-da-Silva, A. Time-dependent and selective microglia-mediated removal of spinal synapses in neuropathic pain. Cell Rep 42(1), 112010, (2023).
4. Favuzzi, E., Huang, S., Saldi, G. A., Binan, L., Ibrahim, L. A., Fernandez-Otero, M., Cao, Y., Zeine, A., Sefah, A., Zheng, K., Xu, Q., Khlestova, E., Farhi, S. L., Bonneau, R., Datta, S. R., Stevens, B. & Fishell, G. GABA-receptive microglia selectively sculpt developing inhibitory circuits. Cell 184, 4048-4063 e4032, (2021).
5. Ruan, C., Sun, L., Kroshilina, A., Beckers, L., De Jager, P., Bradshaw, E. M., Hasson, S. A., Yang, G. & Elyaman, W. A novel Tmem119-tdTomato reporter mouse model for studying microglia in the central nervous system. Brain Behav Immun 83, 180-191, (2020).
6. Mogil, J. S. Animal models of pain: progress and challenges. Nat Rev Neurosci 10, 283-294, (2009).
7. Haring, M., Zeisel, A., Hochgerner, H., Rinwa, P., Jakobsson, J. E. T., Lonnerberg, P., La Manno, G., Sharma, N., Borgius, L., Kiehn, O., Lagerstrom, M. C., Linnarsson, S. & Ernfors, P. Neuronal atlas of the dorsal horn defines its architecture and links sensory input to transcriptional cell types. Nat Neurosci 21, 869-880, (2018).
8. Nicholson, B. & Verma, S. Comorbidities in chronic neuropathic pain. Pain Med 5 Suppl 1, S9-S27, (2004).

Presenting Author

Simran Dhir

Poster Authors

Simran Dhir

McGill University

Lead Author

Rose Rodrigues

PhD

McGill University

Lead Author