Acetylcholine is a neurotransmitter and neuromodulator that critically modulates cognition. We have shown that acetylcholine activates axonal muscarinic receptors in hippocampal dentate gyrus granule neurons to persistently lower the action potential (AP) threshold and enhance neuronal excitability1. This was due to a sustained increase in T-type Ca2+ channel activity resulting in elevated basal Ca2+ levels which inhibited KV7 channels. The intracellular mechanism coupling axonal muscarinic receptors to T-type Ca2+ channels remains unknown. We investigated this by making electrophysiological recordings from hippocampal granule cells present in brain slices obtained from 5-9 week old mice. Cholinergic afferent stimulation or oxotremorine-M (Oxo-M), a muscarinic receptor agonist, application led to a persistent decrease in AP threshold and augmented neuron excitability. Oxo-M treatment also transiently depolarized the resting membrane potential (RMP) and enhanced input resistance (RN). Whilst G-protein inhibition by GDP-bS prevented the RMP depolarization and RN increase by Oxo-M, it had little effect on the sustained AP threshold reduction by Oxo-M or cholinergic afferent stimulation. In support, immunogold labelling with a pan Ga antibody indicated that Ga subunits were absent in axons. Instead, the long-term AP threshold decrease by muscarinic receptor stimulation was prevented by selective knockdown of beta-arrestin 2 in adult granule neurons. Further, two-photon Ca2+ imaging showed that muscarinic receptor activation-induced sustained Ca2+ entry into axons was abolished in β-arrestin 2 knockout neurons. Our findings, thus, strongly suggest that axonal and soma/dendritic muscarinic receptors differentially affect neuronal function by triggering distinct signaling mechanisms.1. Martinello et al., 2015, Neuron, 85, 346-63.