How can I measure brain acetylcholine levels in vivo? Advantages and caveats of commonly used approaches

Abstract

The neurotransmitter acetylcholine (ACh) plays a central role in the regulation of multiple cognitive and behavioral processes, including attention, learning, memory, motivation, anxiety, mood, appetite, and reward. As a result, understanding ACh dynamics in the brain is essential for elucidating the neural mechanisms underlying these processes. In vivo measurements of ACh in the brain have been challenging because of the low concentrations and rapid turnover of this neurotransmitter. Here, we review a number of techniques that have been developed to measure ACh levels in the brain in vivo. We follow this with a deeper focus on use of genetically encoded fluorescent sensors coupled with fiber photometry, an accessible technique that can be used to monitor neurotransmitter release with high temporal resolution and specificity. We conclude with a discussion of methods for analyzing fiber photometry data and their respective advantages and disadvantages. The development of genetically encoded fluorescent ACh sensors is revolutionizing the field of cholinergic signaling, allowing temporally precise measurement of ACh release in awake, behaving animals. Use of these sensors has already begun to contribute to a mechanistic understanding of cholinergic modulation of complex behaviors.image

Unraveling the Neural Circuits: Techniques, Opportunities and Challenges in Epilepsy Research

“…This is attributable to its exceptional sensitivity and high-resolution detection capabilities, especially regarding calcium signal detection in multiple brain regions. However, similar to electrophysiology, fiber photometry faces the problems of low resolution and limited range of brain area monitoring, and its difficulty in controlling the depth of captured signals, which are worthy of subsequent research and improvement (Mineur and Picciotto 2023 ).…”

Cholinergic Mechanisms in Gastrointestinal Neoplasia

“…Studying this is complicated by the constant hydrolysis of ACh by AChE, as well as by the Brownian motion of ACh in the extracellular space (Figure 1). The accuracy of ACh quantification at low concentrations should be an important consideration in experimental design, as ACh concentrations in the peripheral nervous system, including the enteric nervous system of the GI tract, are in the range of 10-100 µM, and even lower in the serosal fluid of the GI tract; the use of supraphysiologic ACh concentrations can be misleading [19,76]. Microdialysis, as described in the previous section, can be used to detect physiological concentrations of ACh.…”

“…After collecting the ACh-containing fluid of interest (which typically will also contain AChE), inhibitors of AChE are added to prevent ACh hydrolysis. While microdialysis can quantify ACh levels in living tissue while filtering out larger molecular weight molecules, excessive addition of AChE inhibitors can skew readouts to falsely high values [76]. High-performance liquid chromatography with electrochemical detection (HPLC-ED) is highly sensitive in detecting ACh from various types of sample preparations (e.g., tissue supernatants, serum, microdialysis samples), with a limit of detection of 5 nM, but it lacks both spatial and temporal resolution and is labor-and time-intensive [74,76].…”

Recent advances in cholinergic mechanisms: A preface for the ISCM2022 special issue

“…Therefore, understanding ACh dynamics in the brain and in vivo measurements of its levels are essential for elucidating the neural mechanisms underlying these processes. Mineur and Picciotto (2023) review a number of techniques that have been developed to measure ACh levels in the brain, including PET, MRS, micro‐dialysis and voltammetry/amperometry, and highlighting the development of genetically encoded fluorescent ACh sensors. The use of such sensors has already begun to contribute to a mechanistic understanding of the cholinergic modulation of complex behaviors, and therefore this method is revolutionizing the field of cholinergic signaling, allowing temporally precise measurements of ACh release in awake, behaving animals (Mineur & Picciotto, 2023).…”