Abstract How our brains identify and respond to speech and other auditory cues remains unclear. Neurons in the inferior colliculus (IC), a hub for auditory processing located in the midbrain, exhibit selective responses to the spectral and temporal features of speech and other complex sounds. Previous findings suggest that acetylcholine (ACh), a neuromodulator associated with attention and synaptic plasticity, may provide an attention-based mechanism to alter auditory processing in the IC. Furthermore, neurons in the IC express different combinations of nicotinic acetylcholine receptor (nAChR) subunits. However, the cellular mechanisms underlying cholinergic modulation in the IC and its impact on downstream targets in the auditory thalamus remain unknown. We recently found that brief pulses of ACh drive prolonged periods of firing in Vasoactive Intestinal Peptide (VIP) neurons in the IC. Moreover, VIP neurons project to the auditory thalamus (medial geniculate, MG), and we obtained preliminary data suggesting that brief pulses of VIP promote strong depolarization in a subset of MG neurons. We hypothesize that ACh enhances excitability of VIP neurons through a nAChR-dependent signaling pathway and that VIP neurons in turn use glutamatergic and VIP signaling to drive prolonged excitability of postsynaptic neurons in the MG. To address this, we are using brain slice electrophysiology and pharmacology to determine the mechanisms that govern the modulatory effects of ACh on VIP neurons and VIP on MG neurons. Our preliminary results show that the prolonged firing of VIP neurons elicited by brief pulses of ACh depends on activation of α4β2 nAChRs. Furthermore, we found that this effect is not abolished by blocking glutamatergic, GABAergic, and glycinergic synaptic transmission or by reducing extracellular Ca2+, suggesting that ACh acts by activating α4β2 nAChRs expressed on VIP neurons themselves and not through activation of neurons presynaptic to VIP neurons. Additionally, our preliminary data show that brief pulses of VIP elicit prolonged depolarization in MG neurons. The overall objective of this proposal is to determine for the first time how cholinergic modulation alters activity in an identified class of IC principal neurons and how these alterations in turn affect activity in the MG. In Aim 1, we will combine brain slice electrophysiology with pharmacology and optogenetics to determine the receptors and mechanisms involved in cholinergic modulation of IC VIP neurons. In Aim 2, we will use brain slice electrophysiology, pharmacology and channelrhodopsin-assisted circuit mapping (CRACM) to determine how synaptic transmission from VIP neurons affects the excitability of MG neurons. Overall, our results will provide a mechanistic basis for how cholinergic modulation in the IC shapes neuron excitability in the IC and MG and will provide a foundation for determining how cholinergic modulation can be used to promote adaptive plasticity in people with h...