Characterisation of a Novel GABA Signalling System in the Human Cerebral Vasculature

Abstract

γ-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the mammalian brain, underlying diverse functions from the maintenance of consciousness and the formation of memories to the regulation of the processes underlying brain development and circadian rhythms. While GABAergic regulation in neuronal tissues has been well characterised, the role of GABA in non-neuronal cell types in the brain has been poorly explored. There is some evidence for the GABAergic regulation of specific vascular functions in the rodent brain and in the human peripheral vasculature, but the receptor systems and cellular machinery underlying this regulation have not been characterised. And as yet, there have been no attempts to confirm the existence of this putative vascular GABA signalling system in the human brain. In this thesis, we present evidence, for the first time, for a novel GABA signalling system in the human cerebral vasculature. Extensive immunohistochemical and immunocytochemical staining studies were conducted in situ in post-mortem tissue from the human middle temporal gyrus (MTG) and in vitro in cultures of human primary MTG pericytes and human cerebral microvascular endothelial cells (hCMVECs), revealing the existence of a unique repertoire of GABAA receptor subunits as well as GABAB receptor subunits and potentially GABA transporters. NanoString nCounter analysis was utilised to confirm the gene expression of GABAA and GABAB receptor subunits by cerebrovascular cells in vitro, and protein expression was confirmed using Western blot. An immunohistochemical study also revealed potential alterations in GABAA receptor subunit densities in Alzheimer’s disease MTG capillaries. ELISA did not reveal the presence of GABA at appreciable concentrations in pericytes or hCMVECs, indicating that these cells likely do not synthesise GABA. Potentiometric dye assays demonstrated that GABA is unlikely to modulate membrane potential in pericytes, while calcium imaging studies demonstrated no modulation of pericytic calcium signalling by GABA receptor agonists. ECIS studies demonstrated that GABA receptor agonists do not play a role in modulation of pericyte contractility. Finally, early studies appear to indicate that ERK1/2 phosphorylation state in pericytes is not regulated by GABAB receptors, but this work is in progress. We conclude that the human cerebral vasculature expresses a GABA signalling system with distinct structural properties, likely indicating a pharmacology unique to the cerebrovascular system. While we were not able to determine the functional relevance of the system, this study lays a foundation for future investigation in this area.