Our ability to think, to memorize and focus our thoughts depends on acetylcholine signaling in the brain. The loss of cholinergic signalling in for instance Alzheimer’s disease strongly compromises these cognitive abilities. The traditional view on the role of cholinergic input to the neocortex is that slowly changing levels of extracellular acetylcholine (ACh) mediate different arousal states. This view has been challenged by recent studies demonstrating that rapid phasic changes in ACh levels at the scale of seconds are correlated with focus of attention, suggesting that these signals may mediate defined cognitive operations. Despite a wealth of anatomical data on the organization of the cholinergic system, very little understanding exists on its functional organization. How the relatively sparse input of cholinergic transmission in the prefrontal cortex elicits such a profound and specific control over attention is unknown. The main objective of this proposal is to develop a causal understanding of how cellular mechanisms of fast acetylcholine signalling are orchestrated during cognitive behaviour.In a series of studies, I have identified several synaptic and cellular mechanisms by which the cholinergic system can alter neuronal circuitry function, both in cortical and subcortical areas. I have used a combination of behavioral, physiological and genetic methods in which I manipulated cholinergic receptor functionality in prefrontal cortex in a subunit specific manner and found that ACh receptors in the prefrontal cortex control attention performance. Recent advances in optogenetic and electrochemical methods now allow to rapidly manipulate and measure acetylcholine levels in freely moving, behaving animals. Using these techniques, I aim to uncover which cholinergic neurons are involved in fast cholinergic signaling during cognition and uncover the underlying neuronal mechanisms that alter prefrontal cortical network function.