A fast genetically encoded fluorescent sensor for faithfulin vivoacetylcholine detection in mice, fish, worms and flies

Abstract

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

Genetically encoded sensors enable micro- and nano-scopic decoding of transmission in healthy and diseased brains

“…However, these sensors may have limited dynamics and/or slow kinetics due to the primogenitors' slow kinetics and limited conformational changes associated with ligand binding [24][25][26]. Because PBPs have lower affinities with their ligands, creating the PBP-based sensors with sensitivity matching the endogenous transmitter levels may require painstaking evolution, yet the effort can lead to high-performance sensors with fast kinetics and large dynamics [27][28][29][30]. Membrane surface trafficking of the PBP-based sensors can be less than optimal, but these sensors are usually more amenable for targeted expression in other subcellular compartments.…”

A Fast High-Affinity Fluorescent Serotonin Biosensor Engineered from a Tick Lipocalin

“…On the other hand, GPCR-based biosensors often respond to all molecules that interact with the parental GPCR, resulting in artifacts when GPCR antagonists or agonists are concurrently used. 11 In addition, GPCR-based biosensors may suffer from slow dissociation kinetics. 11 Furthermore, there are concerns about the overexpression of GPCRs, which may perturb cell signaling that is highly organized and tightly controlled.…”

“…11 In addition, GPCR-based biosensors may suffer from slow dissociation kinetics. 11 Furthermore, there are concerns about the overexpression of GPCRs, which may perturb cell signaling that is highly organized and tightly controlled. 12…”

pMAT: An Open-Source, Modular Software Suite for the Analysis of Fiber Photometry Calcium Imaging

“…Fiber photometry is a tool that allows for the recording of bulk fluorescent signals from a growing number of sensors including those for detecting levels of cellular activity, such as GCaMP6 (Chen et al, 2013), jGCaMP7 (Dana et al, 2019), jRCAMP1a,b or jRGECO1a (Dana et al, 2016). In addition, a number of sensors have recently been developed for detecting the binding of specific neurotransmitters, such as glutamate via iGluSnFr (Marvin et al, 2018), GABA via iGABASnFr (Marvin et al, 2019), acetylcholine via iAChSnFr (Borden et al, 2020), serotonin via iSeroSnFr (Unger et al, 2019), dopamine via dLight (Patriarchi et al, 2018) or GRAB DA (Sun et al, 2018), and norepinephrine via GRAB NE (Feng et al, 2019).…”