Peripheral nerve transection results in the permanent deletion of propriosensory synapses through a Ccl2 mechanism
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Chang, Ethan Y.
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Abstract
Synaptic plasticity has been a topic of great interest, especially given the poor prognosis of motor neuron injuries that result in functional losses. Much research has investigated the effects of peripheral nerve injuries (PNIs) on the degradation of synaptic structures as well as the physiological deficits that result from the injuries (Alvarez et al., 2011; Bullinger et al., 2011). Unfortunately, not much is known about the mechanisms of how synaptic plasticity occurs following PNI and how that may contribute to motor deficits observed in injured models. This paper attempts to explore Ccl2, a ligand implicated in the recruitment of Ccr2 immune cells, as a possible intermediate involved in the removal VGluT1, a glutamate transporter used to assess Ia afferent synaptic health, following a nerve transection cut-repair procedure (Rotterman et al., 2019; Alvarez et al., 2011). The paper also attempts to determine if blocking the Ccl2-Ccr2 mechanism results in restoration of Ia synaptic contacts. 4 groups of 4-6 mice were used in the experiment: a control with no genetic alteration, Ccl2^(flx/flx) group as a genetic control, 〖MN〗^ΔCcl2 experimental group with Ccl2 specifically KO in motoneurons, and 〖MG〗^ΔCcl2 experimental group with Ccl2 specifically KO in microglia. Ccl2-mCherryflox mice used in the experiment allowed for visualization of the presence or absence of Ccl2 via the fluorescent reporter protein mCherry. The fluorescent retrograde enhancer Fast Blue was administered to label motoneurons including their dendritic branches, and immunohistochemistry was performed to label VGluT1. Confocal z-stack images were then used to generate 3D reconstructions utilized in the determination of VGluT1 density following nerve cut and repair. A one-way ANOVA and post-hoc Bonferroni determined significant decreases (p<0.05) in VGluT1 density on the dendrites and somas of Ccl2^(flx/flx) (32.5% and 48.7% respectively) and 〖MN〗^ΔCcl2 (26.7% and 42.0% respectively) compared to control, but 〖MG〗^ΔCcl2 groups only demonstrated a significant decrease in VGluT1 density (37.2%) on the soma. These results are compounded with the analysis that 75.3% ± 3% (±SD) of VGluT1 synapses are located on dendrites. As a result, it was concluded that selective KO of VGluT1 on microglia in the 〖MG〗^ΔCcl2 group recued VGluT1. Although Ccl2 was identified as one possible agent in the degradation pathway of VGluT1 and thus implicated in the synaptic plasticity changes following nerve cut, it remains unclear what agents are involved in the actual removal of the synaptic structures. Future studies can further explore, which specific agents are recruited by Ccl2 signaling as well as functional differences that result despite structural recovery.
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