A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies

Jing, Miao; Zhang, Peng; Wang, Guangfu; Feng, Jiguang; Mesik, Lukas; Zeng, Jianzhi; Jiang, Hua; Wang, Shaohua; Looby, Jess C; Guagliardo, Nick A.; Langma, Linda W; Ju, Lu; Zuo, Yi; Talmage, David A.; Role, Lorna W.; Barrett, Paula Q.; Zhang, Li I.; Luo, Minmin; Song, Yan; Zhu, Jie; Li, Yulong

doi:10.1038/nbt.4184

Cited by 304 publications

(304 citation statements)

References 77 publications

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“…Importantly, we utilized contralateral fiber implants and red-shifted geneticallyencoded calcium indicators for the VTA in order to exclude the possibility of picking up spurious signals from VTA axons projecting to the ventral striatum. For measurement of 35 extracellular acetylcholine, wild type mice were bilaterally injected with AAV ACh3.0, an improved variant of a recently described fluorescent acetylcholine sensor (26).…”

Section: Viral Injection and Fiber Implantationmentioning

confidence: 99%

Basal forebrain-derived acetylcholine encodes valence-free reinforcement prediction error

Sturgill

Hegedűs

et al. 2020

Preprint

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Acetylcholine (Ach) is released by the cholinergic basal forebrain (CBF) throughout the cortical mantle and is implicated in behavioral functions ranging from arousal to attention to learning. Yet what signal ACh provides to cortex remains unresolved, hindering our understanding of its functional roles. Here we demonstrate that the CBF signals unsigned reinforcement prediction error, in contrast to dopamine (DA) neurons that encode reward 25 prediction error. We show that both CBF neuronal activity and acetylcholine (ACh) release at cortical targets signal reinforcement delivery, acquire responses to predictive stimuli and show diminished responses to expected outcomes, hallmarks of a prediction error. To compare ACh with DA, we simultaneously monitored the activity of both neuromodulators during a serial reversal learning task. ACh tracked learning as swiftly as DA during acquisition but lagged 30 slightly during extinction, suggesting that these neuromodulators play complementary roles in reinforcement as their patterns of innervation, cellular targets, and signaling mechanisms are themselves complementary. Through retrograde viral tracing we show that the cholinergic and dopaminergic systems engage overlapping upstream circuits, accounting for their coordination during learning. This predictive and valence-free signal explains how ACh can proactively and 35 retroactively improve the processing of behaviorally important stimuli, be they good or bad.

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Section: Viral Injection and Fiber Implantationmentioning

confidence: 99%

Basal forebrain-derived acetylcholine encodes valence-free reinforcement prediction error

Sturgill

Hegedűs

et al. 2020

Preprint

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“…Protein-based biosensors allow targeted expression in cells, and even sub-cellular compartments, of interest. A recently published sensor (GACh2.0) 20 couples ACh binding to a change in the fluorescence of circularly permuted green fluorescent protein (cpGFP) inserted within an intracellular loop of the M3 mAChR. GACh2.0 has an apparent ACh affinity of 700 nM, a maximal fluorescence increase of ~80% in cultured cells, and kinetics of ~200 ms trise and ~700 ms tdecay 20 .…”

mentioning

confidence: 99%

“…However, molecular characteristics of the GACh2.0 sensor restrict its utility. Foremost, in flies and mice GACh2.0 requires seconds for the onset and decay of ACh responses 20 . This precludes resolution of faster signaling events, hinders detection of sparse events, and thus renders GACh2.0 essentially a binary detector of ACh within a region of interest over several seconds.…”

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confidence: 99%

A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies

Zhang

Shivange

et al. 2020

Preprint

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138

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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

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“…We proceeded to examine the dynamics of active CB 1 upon the CB 1 agonist, WIN 55,212-2 (WIN), application using live SIM imaging. Previously, several probes have been developed to detect neuromodulator receptor activations, such as dopamine (DA), norepinephrine (NE), and acetylcholine (Ach) 22,23,24 . An agonist-induced conformational change of these GPCRs alters the arrangement of the associated cpGFP and results in a fluorescence change.…”

Section: Active Cb 1 Tightly Associate With Mps and Display Less Dynamentioning

confidence: 99%

Organized cannabinoid receptor distribution in neurons revealed by super-resolution fluorescence imaging

Yang

Tian

et al. 2020

Preprint

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G-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB 1 ), we discovered periodically repeating clusters of CB1 hotspots within the axons of neurons. We observed these CB 1 hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB 1 signaling. Furthermore, we found that CB 1 hotspot periodicity increased upon CB 1 agonist application, and these activated CB1 displayed less dynamic movement compared to non-activated CB 1 . Our results suggest that CB 1 forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy when being activated.

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A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies

Cited by 304 publications

References 77 publications

Basal forebrain-derived acetylcholine encodes valence-free reinforcement prediction error

Basal forebrain-derived acetylcholine encodes valence-free reinforcement prediction error

A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies

Organized cannabinoid receptor distribution in neurons revealed by super-resolution fluorescence imaging

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