The ability to directly measure acetylcholine (ACh) release is an essential step towards understanding its physiological function. Here, we optimized the GRAB ACh ( G PC R - A ctivation‒ B ased- ACh ) sensor to achieve substantially improved sensitivity in ACh detection, as well as reduced downstream coupling to intracellular pathways. The improved version of the ACh sensor retains the sub-second response kinetics, physiological-relevant affinity and precise molecular specificity to ACh of its predecessor. Using this sensor, we revealed compartmental ACh signals in the olfactory center of transgenic flies in response to external stimuli including odor and body shock. Using fiber photometry recording and two-photon imaging, our ACh sensor also enabled sensitive detection of single-trial ACh dynamics in multiple brain regions in mice in a variety of behaviors.

The ability to directly measure acetylcholine (ACh) release is an essential first step towards understanding its physiological function. Here we optimized the GRABACh (GPCR-Activation-Based-ACh) sensor with significantly improved sensitivity and minimal downstream coupling. Using this sensor, we measured in-vivo cholinergic activity in both Drosophila and mice, revealing compartmental ACh signals in fly olfactory center and single-trial ACh dynamics in multiple regions of the mice brain under a variety of different behaviors 2 / 37 Cholinergic signals mediated by the neurotransmitter ACh are involved in a wide range of physiological processes, including muscle contraction, cardiovascular function, neural plasticity, attention and memory 1-3 .Previously, cholinergic activity was mainly measured using either electrophysiology to record nicotinic receptormediated currents 4, 5 or microdialysis followed by biochemical purification and identification 6 . However, these methods generally lack both cell-type specificity and the spatial-temporal resolution needed to precisely dissect cholinergic signals in vivo. Combining the type 3 muscarinic ACh receptor (M3R) with the conformationalsensitive circular permutated GFP (cpGFP), we recently developed GACh2.0 (short as ACh2.0), a genetically encoded GRAB (GPCR-Activation Based) ACh sensor that can convert the ACh-induced conformational change on M3R into a sensitive fluorescence response 7 . The ACh2.0 sensor responds selectively to physiological concentration of ACh with an EC50 of 2 μM and has been used in several model organisms to detect the endogenous release and regulation of cholinergic signals. Here, we optimized the GRABACh sensor using sitedirected mutagenesis and cell-based screening to further increase the sensitivity.To improve the performance of the GRABACh sensor, we focused on the interface between M3R and cpGFP, including the receptor's third intracellular loop (ICL3) and linker peptides, as well as critical residues in cpGFP that contribute to its fluorescence intensity (Figs. 1A and S1A-D). Our initial screening based on mediumthroughput imaging identified several variants with improved performance; these variants were subsequently verified using confocal microscopy (see Methods for details). The sensor with the largest ACh-induced fluorescence response was selected for further study and is named as GRABACh3.0 or ACh3.0 (Fig. 1A). We also generated a ligand-insensitive form of ACh3.0 by introducing the W200A mutation into the receptor 8 (Figs. 1A and S1E). When expressed in HEK293T cells or cultured neurons, the ACh3.0 sensor localized to the plasma membrane of the soma, and trafficked to dendrites and axons in neurons ( Fig. 1B-D). Moreover, compared to ACh2.0, the ACh3.0 sensor had a significantly larger fluorescence change (ΔF/F0~280%) in response to 100 μM ACh (Figs. 1B-D and S2A-E); in contrast, the ligand-insensitive ACh3.0-mut sensor had no detectable 4 coverslips for ACh2.0, ACh3.0, and ACh3.0-mut, respectively, with an average of >20 cells per co...

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Postural instability and gait disturbances, common disabilities in the elderly and frequently present in Parkinson's disease (PD), have been suggested to be related to dysfunctional cholinergic signaling in the brainstem. We investigated how long-term loss of cholinergic signaling from mesopontine nuclei influence motor behaviors. We selectively eliminated the vesicular acetylcholine transporter (VAChT) in pedunculopontine and laterodorsal tegmental nuclei cholinergic neurons to generate mice with selective mesopontine cholinergic deficiency (VAChT ). VAChT mice did not show any gross health or neuromuscular abnormality on metabolic cages, wire-hang and grip-force tests. Young VAChT mice (2-5 months-old) presented motor learning/coordination deficits on the rotarod; moved slower, and had smaller steps on the catwalk, but showed no difference in locomotor activity on the open field. Old VAChT mice (13-16 months-old) showed more pronounced motor learning/balance deficits on the rotarod, and more pronounced balance deficits on the catwalk. Furthermore, old mutants moved faster than controls, but with similar step length. Additionally, old VAChT-deficient mice were hyperactive. These results suggest that dysfunction of cholinergic neurons from mesopontine nuclei, which is commonly seen in PD, has causal roles in motor functions. Prevention of mesopontine cholinergic failure may help to prevent/improve postural instability and falls in PD patients. Read the Editorial Highlight for this article on page 688.

It is well established that neurons secrete neuropeptides and ATP with classical neurotransmitters; however, certain neuronal populations are also capable of releasing two classical neurotransmitters by a process named co-transmission. Although there has been progress in our understanding of the molecular mechanism underlying co-transmission, the individual regulation of neurotransmitter secretion and the functional significance of this neuronal 'bilingualism' is still unknown. Striatal cholinergic interneurons (CINs) have been shown to secrete glutamate (Glu) in addition to acetylcholine (ACh) and are recognized for their role in the regulation of striatal circuits and behavior. Our review highlights the recent research into identifying mechanisms that regulate the secretion and function of Glu and ACh released by CINs and the roles these neurons play in regulating dopamine secretion and striatal activity. In particular, we focus on how the transporters for ACh (VAChT) and Glu (VGLUT3) influence the storage of neurotransmitters in CINs. We further discuss how these individual neurotransmitters regulate striatal computation and distinct aspects of behavior that are regulated by the striatum. We suggest that understanding the distinct and complementary functional roles of these two neurotransmitters may prove beneficial in the development of therapies for Parkinson's disease and addiction. Overall, understanding how Glu and ACh secreted by CINs impacts striatal activity may provide insight into how different populations of 'bilingual' neurons are able to develop sophisticated regulation of their targets by interacting with multiple receptors but also by regulating each other's vesicular storage.