PROJECT SUMMARY Acetylcholine critically controls complex neurological functions through its modulation of brain circuits. In contrast to the well-studied mechanisms of acetylcholine signaling at the neuromuscular junction, its transmission modes and mechanisms in the vertebrate central nervous system are not well understood. In the striatum, acetylcholine exerts rapid and powerful control over local dopaminergic activity. Together, these neuromodulators regulate a variety of important behaviors, including motivation and reward-related learning. Because acetylcholine is rapidly degraded after it is released in striatal tissues, the fidelity of acetylcholine to dopamine signaling must involve either tight spatial relationships between release sites and receptors, and/or fast transmission. However, neuromodulator signaling is classically modeled as occurring through volume transmission, involving dispersed, non-specific release as opposed to synaptic, point-to-point signaling – a concept that remains to be proven. Furthermore, the molecular mechanisms underlying striatal acetylcholine signaling remain elusive. I hypothesize that sparse cholinergic terminals require molecular elements for highly synchronous acetylcholine release. In this scenario, rapid and precise vesicle release could produce a synchronous wave of high-concentration acetylcholine, which might allow even distant receptors to sense this signal with potency. To test this hypothesis, I propose to examine the morphological and molecular substrate of striatal acetylcholine transmission in two aims. First, I will dissect the structure of acetylcholine to dopamine signaling in the striatum, including the presence of secretory machinery in acetylcholine nerve terminals and their apposition to acetylcholine receptors on dopamine axons, using superresolution microscopy. In a second aim, I will functionally test whether acetylcholine release onto dopamine axons requires secretory proteins that are predestined to generate fast and precise release, enabling phasic acetylcholine transmission. Additionally, I will determine how this transmission mode impacts subsequent dopamine signaling. To do this, I will use genetic tools paired with simultaneous imaging of fluorescent acetylcholine sensors and amperometric dopamine measurements in striatal slices. Together, these findings will inform our fundamental understanding of cholinergic to dopaminergic signaling in the striatum, including the identification of important genes that regulate acetylcholine transmission. This molecular-level understanding will ultimately be important to better understand brain function and disease.

PROJECT NARRATIVE Neuromodulatory signaling in the brain provides critical control of a variety of complex behaviors including motivation, reward-learning, and locomotion. Acetylcholine is an important modulatory transmitter in the striatum, a brain structure that controls these functions. The proposed work aims to dissect the molecular mechanisms of acetylcholine transmission in the striatum, with the overarching goal to better understand neuromodulatory transmission in health and disease.