Glutamatergic signaling after rachimedullary trauma: role of glial cells in the inflammatory response and excitotoxicity

Abstract

Proximal axotomies are especially severe, leading to up to 50% motoneuron degeneration in the first few weeks, in the case of ventral root crush (VRC). Microglia are the first cells to respond to injury with activation and migration towards the injured motoneurons. Astrocytes also respond through morphofunctional changes, collectively called reactive astrogliosis. Recent evidence indicates that glutamatergic signaling, via AMPA-type receptors, may be related to neuroprotective effects after rachimedulary injuries. Therefore, the present project aims to understand the signaling mechanisms between the injured motoneuron, via AMPA receptors, after the murine model of VRC, and the surrounding glia. To this end, initially a temporal study of the expression of AMPA receptors in glial cells will be carried out (by immunohistochemistry), which will be correlated with the curve of motoneurons loss (by Nissl-staining). Next, the pharmacological blockade of AMPA receptors will be performed after VRC, for a comparative study of glial reaction and neuronal survival. Furthermore, we will study how a known neuroprotective treatment, the cannabidiol, modulates the glial expression of AMPA receptors, and correlate with the results obtained previously. To specifically study the role of astrocytic AMPA receptors in the context of neuronal injury and death, conditional knockout animals (cKO, GFAP-CreERT2::GluA1fl/fl::GluA4fl/fl::R26-tdT) will be used. All animals will undergo motor function recovery analysis through the Catwalk system, as well as electrophysiological analysis to assess muscle reinnervation. Finally, in collaboration with researchers from the Saarland University, Germany, we will study in vivo, through two-photon laser microscopy (2P-LSM), the morphofunctional changes in glial cells after injury, using the cko and transgenic animals expressing fluorescent proteins in specific cell types, as well as the pattern of generation and propagation of calcium waves in astrocytes, through the specific expression of the GCaMP3 reporter. (AU)