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.