Although there is considerable evidence for the actions of ACh on target neurons, the mode of cholinergic transmission has remained controversial. The debate has focused on whether cholinergic signaling occurs via traditional synapses (cellular specializations comprising closely apposed pre- and postsynaptic membranes with associated release/receptor machinery) or via volume transmission (actions of a neurotransmitter that occur at a distance from its site of release, mediated by diffusion through the extracellular space (Zoli et al., 1999). Accumulating evidence indicates that ACh can act through volume transmission in the brain. The relatively diffuse nature of brain cholinergic innervation further reinforces this idea. There is an anatomical mismatch between the sites of ACh release (Houser, 1990; Wainer et al., 1984a; Wainer et al., 1984b) and the location of cholinergic receptors (Arroyo-Jimenez et al., 1999; Hill Jr. et al., 1993; Kawai et al., 2007). There is also evidence that extracellular levels of ACh fluctuate in a manner that is not consistent with localized clearance of a synaptic transmitter (Hajnal et al., 1998; Laplante et al., 2004; Mark et al., 1996; Parikh et al., 2004;
