Neuron-glia interactions and neuroinflammation: in vitro approaches to elucidate spinal cord disorders pathophysiology

Neuroinflammation is a characterizing trait of various central nervous system (CNS) pathologies, from neurodegenerative diseases to neuropsychiatric disorders. In the effort of dissecting the impact of immune status alterations on neural circuit function, we focused our study on the effects of local inflammation in a controlled micro-environment where neurons and neuroglial cells maintain their appropriate organization: the organotypic spinal cord slices. These cultures, developed from the spinal cord of mouse embryos, represent a complex in vitro model where sensory-motor cytoarchitecture, synaptic properties and spinal cord resident cells, encompassing heterogeneous neuronal phenotypes and neuroglia, are retained in a 3D fashion. I have used this model to mainly investigate: 1) synaptic spinal changes induced by inflammation and mechanisms by which the inhibitory transmission is modulated; 2) resident microglia and astrocytes reactivity to inflammatory stress; 3) astrocytes calcium activity tuned by inflammation; 4) mechanisms which modulate calcium release and spreading in the inflamed tissues. Overall, my main aim was to understand the link between neuroinflammation and spinal pre-motor network. For the purpose, organotypic spinal cord slices are cultured for two weeks in vitro. Then, they are exposed for 4 and 6 hours to a cocktail of cytokines (CKs, 10 ng/mL), composed by tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta) and granulocyte macrophage-colony stimulating factor (GM-CSF), or to lipopolysaccharide (LPS, 1 µg/mL). I use single cell electrophysiology, live cell calcium imaging, immunocytochemistry, confocal microscopy and immunoblotting analysis to investigate and compare the spinal tissue responses to neuroinflammation induced by CKs and LPS. I first focus on the synaptic level, describing a progressive increase in the frequency of spontaneous post-synaptic currents (sPSCs) and inhibitory post-synaptic currents (IPSCs) and the shortening of GABAergic current due to CKs incubations, absent in LPS treated ones. I further explore by immunofluorescence and confocal microscopy, resident neuroglia reactivity, describing the activation of different inflammatory status, given by the two different paradigms, confirmed by the presence of cytokines and chemokines released in the supernatants. By live calcium imaging, I document an increase in the occurrence of calcium oscillations displayed by the astrocytes located in the ventral horn, and by several pharmacological treatments, I dissect the behavior of the oscillations, trying to understand their origin and mechanism of spreading. In conclusion, my work compared the effects of a cytokines cocktail and of LPS in altering the pre-motor circuits signaling, that may contribute to sustain spinal network increased excitability, eventually leading to neurodegeneration.