New insight into the role of adenosine and acetylcholine receptors on neuronal excitability and oligodendrogliogenesis: an in vitro study

In the CNS, acetylcholine (ACh) is a key neurotransmitter implicated in higher brain functions, including cognitive processes. The Nucleus Basalis of Meynert (NBM) is the major source of cholinergic input to the cerebral cortex and projects to most cortical areas and is implicated in several roles, as memory, attention, and behaviour. It has been demonstrated that the degeneration of NBM is involved in various forms of dementia, as Alzheimer and Parkinson diseases, but also in schizophrenia. The first aim of this thesis was to took advantage from the availability of human foetal nucleus basalis of Meynert (hfNBM) cultures to investigate the electrophysiological properties of immature, non-differentiating, cholinergic neurons from the human developing CNS and their functional responses to cholinergic agonists. For this purpose, we used electrophysiological patch-clamp recordings and selective cholinergic agonist and/or antagonist, to investigate functional metabotropic receptors in hfNBM cultures. Carbachol activated atropine-sensitive muscarinic receptors that enhanced IK and reduced INa currents by activating Gi- or phospholipase C-dependent pathways, respectively. When investigated in the current-clamp mode, cells presented either a single, small amplitude action potential, or high-frequency oscillations in membrane potential. This latter phenomenon, defined by us “voltage waves”, was impaired by intracellular thapsigargin, a potent inhibitor of endoplasmic reticulum Ca++-ATPases, or BAPTA and prevented by extracellular Iberiotoxin, a selective blocker of BK channel, or Ba++, demonstrating the involvement of intracellular Ca++ rise, BK and Kir channels, respectively. This knowledge could be of relevance to understand the mechanisms of cholinergic system development and functions in the human brain, either in health or disease. In addition, it is well recognised that purinergic signalling plays a fundamental role in several biological systems, including both short-term (neurotransmission, endothelial-mediated vasodilatation, platelet aggregation) and long-term (cell proliferation, differentiation, migration and death) phenomenon. In particular, about this field, we studied the role of adenosine in oligodendrogliogenesis and pain. Oligodendrocytes are the only myelinating cells in the brain and differentiate from their progenitors (OPCs) throughout adult life. However, this process fails in demyelinating pathologies. Adenosine is emerging as an important player in OPC differentiation and it is demonstrated that adenosine A2A receptors inhibit cell maturation by reducing voltage-dependent K+ currents. Therefore, no data are available to date about the A2B receptor (A2BR) subtype. On the other hand, the bioactive lipid mediator sphingosine-1-phosphate (S1P) and its receptors (S1P1–5) are also crucial modulators of OPC development. In addition, an interaction between this pathway and the A2BR is reported in peripheral cells. Therefore, the second aim was to study the role of A2BRs in modulating K+ currents and cell differentiation in OPC cultures and we investigated a possible interplay with S1P signalling by electrophysiological recordings and biochemical assays, as real-time quantitative polymerase chain reaction experiments (RT-PCR), western-blot, small interference RNA transfection and immunofluorescence analysis. Our data indicate that the A2BR agonist BAY60-6583 and its new analogue P453 inhibit K+ currents in cultured OPC. This effect was prevented by the A2BR antagonist MRS1706, by K+ channel blockers and was differently modulated by the S1P analogue FTY720-P. An acute (10 min) exposure of OPCs to BAY60-6583 also increased the phosphorylated form of sphingosine kinase 1 (SphK1). A chronic treatment with A2BR agonists decreased OPC differentiation whereas SphK1/2 inhibition exerted the opposite effect. Furthermore, A2BR was overexpressed during OPC differentiation, an effect prevented by the pan SphK1/2 inhibitor VPC69047. Finally, A2BR silenced cells showed increased cell maturation, decreased SphK1 expression and enhanced S1P lyase levels. In conclusion, A2BRs inhibit K+ currents and cell differentiation and positively modulate S1P synthesis in cultured OPCs. Recently, studies have focused on the antihyperalgesic activity of the A3 adenosine receptor (A3R) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Therefore, in the last aim, we investigated the expression and functional effects of A3R on the excitability of small- to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats, by using patch-clamp and RT-PCR experiments and immunofluorescence analysis. Patch-clamp experiments demonstrated that two distinct A3R agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca++-activated K+ (KCa) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca++ influx via N-type voltage-dependent Ca++ channels, as Cl-IB-MECA–induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca++ currents, and its effect was inhibited by 56% in the presence of A3R antagonist MRS1523, demonstrating that the majority of adenosine’s effect is mediated by this receptor subtype. Furthermore, current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A3R activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca++ measurements confirmed the inhibitory role of A3R on DRG neuronal firing. We conclude that pain-relieving effects observed on A3R activation could be mediated through N-type Ca++ channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A3R-based therapy as a viable approach to alleviate pain in different pathologies.