Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals. IntroductionAcetylcholine (ACh) is a critical neurotransmitter in all animals. Among invertebrates, it is the most prevalent excitatory transmitter in the brain, sensory ganglia, and frequently the neuromuscular junction (NMJ). Among vertebrates, only a minority of neurons release ACh, but these signals play varying key roles. For instance, ACh signals at the NMJ, in the autonomic nervous system, and in subsets of the central nervous system, particularly projections arising from the brainstem and basal forebrain. Other cholinergic neuron populations in the brain include striatal interneurons, the stria vascularis-medial habenula-interpeduncular nucleus pathway, and sparse, incompletely characterized cell types such as intrinsic cholinergic interneurons in cortex 1 and hippocampus 2 . ACh helps to regulate attention 3 and wakefulness 4 , and participates in memory formation and consolidation 5 . ACh is also an important transmitter in glia, and between the nervous and immune systems 6 .Acetylcholine is synthesized pre-synaptically from choline and acetyl-CoA by choline acetyltransferase (ChAT), then packaged into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). A key, partially understood aspect of cholinergic signaling is co-release with other neurotransmitters, including GABA, ATP, and glutamate 7,8 . To understand the role of co-release, one must measure ACh release alongside emerging measurements of other neurotransmitters.Acetylcholine receptors are among the most diverse neurotransmitter receptor families. Humans possess five muscarinic G protein-coupled receptors (GPCRs) for ACh (mAChRs) with diverse expression in the brain and smooth, cardiac, and skeletal muscle. Vertebrate nicotinic ACh receptors (nAChRs) are pentameric ligand-gated cation channels. Humans have a total of 17 nAChR subunit genes, in five classes: 10 a, 4 b, and one each of g, d, and e. nAChRs occur with many subunit combinations 9 , and others may be undiscovered. Invertebrates also have AChgated chloride channels. On neurons, receptors can be localized pre-, post-, and extrasynaptically, often with different isoforms in each place 10
Acetylcholine (ACh) regulates a diverse array of physiological processes throughout the body, yet cholinergic transmission in the majority of tissues/organs remains poorly understood due primarily to the limitations of available ACh-monitoring techniques. We developed a family of Gprotein-coupled receptor activation-based ACh sensors (GACh) with sensitivity, specificity, signal-to-noise ratio, kinetics and photostability suitable for monitoring ACh signals in vitro and in vivo. GACh sensors were validated with transfection, viral and/or transgenic expression in a dozen types of neuronal and non-neuronal cells prepared from several animal species. In all preparations, GACh sensors selectively responded to exogenous and/or endogenous ACh with robust fluorescence signals that were captured by epifluorescent, confocal and/or two-photon microscopy. Moreover, analysis of endogenous ACh release revealed firing pattern-dependent release and restricted volume transmission, resolving two long-standing questions about central cholinergic transmission. Thus, GACh sensors provide a user-friendly, broadly applicable toolbox for monitoring cholinergic transmission underlying diverse biological processes.Acetylcholine (ACh), the first identified neurotransmitter, mediates cell-to-cell communication in the central and peripheral nervous systems, as well as non-neuronal systems 1-7 . Cholinergic projection neurons within the mammalian brain primarily originate in three major nuclear groups 1-5 , including the basal forebrain nuclei (BF), and the brainstem pedunculopontine and laterodorsal tegmental nuclei.Cholinergic neurons within these groups project widely throughout the cortical and subcortical domains, consistent with their involvement in complex brain functions, including attention, perception, associative learning and sleep/awake states. Additional smaller populations of cholinergic neurons scatter throughout other brain areas (e.g., the medial habenula (MHb) and the striatum), contributing to behaviors related to motion, motivation and stress 1, 3, 8 . Dysregulation of central cholinergic transmission is linked to a number of brain disorders, including Alzheimer's disease, addiction, epilepsy, Parkinson's disease, schizophrenia and depression. In the peripheral nervous and non-nervous systems, ACh is released by both neurons and non-neuronal cells to relay fast transmission at the neuromuscular junction and to regulate functions of a variety of other tissues and organs, including the heart, liver and pancreas 5-7 . Dysregulation of peripheral and non-neuronal cholinergic signals is associated with multiple pathological states, including cardiovascular disease, obesity, diabetes, immune deficiency and cancer.Despite the significance of ACh signals in many, fundamental aspects of our physiology, cholinergic transmission in the majority of tissues and organs remain poorly understood, due primarily to the limitations of tools available for the direct measurement of ACh 1, 5, 9 . Microdialysis, an established method for monitor...
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