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

Potassium ion (K+) homeostasis and dynamics play critical roles in biological activities. Here we describe three genetically encoded K+ indicators. KIRIN1 (potassium (K) ion ratiometric indicator) and KIRIN1-GR are Förster resonance energy transfer (FRET)-based indicators with a bacterial K+ binding protein (Kbp) inserting between the fluorescent protein FRET pairs mCerulean3/cp173Venus and Clover/mRuby2, respectively. GINKO1 (green indicator of K+ for optical imaging) is a single fluorescent protein-based K+ indicator constructed by insertion of Kbp into enhanced green fluorescent protein (EGFP). These indicators are suitable for detecting K+ at physiologically relevant concentrations in vitro and in cells. KIRIN1 enabled imaging of cytosolic K+ depletion in live cells and K+ efflux and reuptake in cultured neurons. GINKO1, in conjunction with red fluorescent Ca2+ indicator, enable dual-color imaging of K+ and Ca2+ dynamics in neurons and glial cells. These results demonstrate that KIRIN1 and GINKO1 are useful tools for imaging intracellular K+ dynamics.

Near-infrared (NIR) genetically encoded calcium ion (Ca2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross talk with visible light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report 2 improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and Caenorhabditis elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca2+ dynamics.