The working hypothesis is that any stimulus acting on brain microvascular endothelium, might activate the neurovascular unit by intercellular crosstalk, and consequently could increase neuronal hyperexcitability. The first chapter presents the concept of neurovascular unit. Interactions between neurovascular unit components are also discussed, and their involvement in ictogenesis. The second chapter describes the aim of the thesis, the main objectives and the principal technical approaches used in the study. The third chapter is dedicated to the elusive interaction between Qtracker\uae800 vascular labels and brain endothelial microvascular endothelial cells. Results: (1) non-targeted PEGylated near-infrared emitting Qtracker\uae800 accumulate in brain vascular endothelium, (2) Qtracker\uae800 alters \u2018normal\u2019 calcium signalling in brain endothelial cells, (3) there are substantial inter-individual differences in human endothelial cells activation upon exposure to Qtracker\uae800, (4) Qtracker\uae800 may not be suitable for translational studies. In conclusion, although brain vasculature imaging techniques can greatly benefit from the use of nanoparticle labels, such labels may be internalized by and functionally interact with blood vessel endothelia, which raises obvious safety concerns. The fourth chapter makes a functional description of the muscarinic acetylcholine receptors in brain microvascular endothelial cells. Results: (1) all muscarinic acetylcholine receptors (M1-M5) are expressed in mouse brain microvascular endothelial cells, (2) acetylcholine activates calcium transients in brain endothelium via muscarinic, but not nicotinic, receptors, (3) The relative mRNA expression of M2-M5 correlates with their relative protein abundance, but a mismatch exists for M1 mRNA versus protein levels, (4) although M1 and M3 are the most abundant receptors, only a small fraction of the M1 is present in the plasma membrane and functions in ACh-induced Ca2+ signaling, (5) bioinformatic analysis performed on eucaryotic species demonstrate the high degree of conservation of the orthosteric binding site and the great variability of the allosteric site, (6) muscarinic acetylcholine receptors represent potential pharmacological targets in future translational studies. In conclusion, our findings indicate that investigators should particularly focus on the allosteric binding sites of the M1 and M3 receptors. The fifth chapter describes molecular mechanisms of pilocarpine action at the level of brain microvascular endothelium. Results: (1) pilocarpine induces the in vivo and in vitro increase of the cytokines levels, (2) pilocarpine upregulates the expression of adhesion molecules in brain microvascular endothelial cells, (3) pilocarpine elicits calcium transients in brain microvascular endothelial cells but is not inducing epileptic-like discharges in hippocampal pyramidal neurons, (4) pilocarpine downregulates the expression of tight junction proteins and permeabilizes the monolayers of brain microvascular endothelial cells, (5) pilocarpine competes with acetylcholine on the same binding site of the muscarinic receptors and regulates their expression. In conclusion, our study indicates that brain endothelium is an important site of action for pilocarpine, and that neurons\u2019 exposure to pilocarpine is not triggering seizure-like activity, therefore epileptogenesis mechanisms should be revisited
