Protocol to visualize the distribution of exogenously administered small molecules in the mouse brain

Here, we present fixation-driven chemical crosslinking of exogenous ligands, a protocol to visualize the distribution of exogenously administered small molecules in the mouse brain. We first describe the probe design of the small molecules of interest and the probe microinjection into a live mouse brain in detail. We then detail procedures for paraformaldehyde-perfusion fixation. This approach is especially useful for imaging-based evaluation of the small-molecule ligands distribution in mouse brain tissue relying on their interaction with endogenous proteins

Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains

生きた動物脳内で発現する神経伝達物質受容体に目印を付ける新手法を開発 --遺伝子操作を伴わず、生体内でたんぱく質の機能解析が可能に--. 京都大学プレスリリース. 2024-02-05.Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse–chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions

Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution changes

ホルマリン漬けから着想した小分子可視化法 --医薬品開発効率化につながる新たな戦略--. 京都大学プレスリリース. 2022-12-05.Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/excretion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein, we report a new method termed “fixation-driven chemical cross-linking of exogenous ligands (FixEL), ” which traps and images exogenously administered molecules of interest (MOIs) in complex tissues. This method relies on protein-MOI interactions and chemical cross-linking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOIs such as the scaffold of PET tracer derivatives or drug-like small molecules