Chemical labelling for visualizing native AMPA receptors in live neurons

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders.


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Representative confocal images are shown. In f, the cells were stained with a streptavidin-Ax555 conjugate (SAv-Ax555) after labeling with CAM2(Bt). In g, the cells labeled with CAM2(CypHer) in the absence of NBQX were imaged in MES buffered saline at pH 5.5 (20 mM MES, 107 mM NaCl, 6 mM KCl, 2 mM CaCl 2 , and 1.2 mM MgSO 4 ) (in top) or HBS at pH 7.4 (in middle). This indicates that labeled CypHer shows high fluorescence under acidic conditions. EGFP or mCherry was utilized as a transfection marker. Scale bars, 10 µm. of CAM2(Ax488), the cells were incubated for 2 h at 37 °C to promote internalization S15 of labeled GluA2. Labeled GluA2 were observed mainly from intracellular regions and partly from cell surface (left). TB treatment failed to quench intracellular fluorescence, although fluorescence from cell-surface was quenched in this condition.
(right). Scale bar, 5 µm. (c) Comparison of quenching ratio (I TB /I initial ) obtained from surface-exposed GluA2 (n = 6) and internalized GluA2 (n = 7) as shown in b. This indicates that TB treatment selectively quenches the fluorescence of surface-exposed labeled GluA2. (d) Cultured hippocampal neurons labeled by 1 µM of CAM2(Ax488) were subjected to TB treatment. Orange square ROIs indicated in upper panels are expanded in lower panels. Scale bars, 10 µm in upper panels and 5 µm in lower panels.
(e) Comparison of I TB /I initial obtained from hippocampal neurons (in d, n = 12) and HEK293T cells (in b, n = 6). Student's t-test indicates that significant differences were not observed, which indicates that surface-exposed AMPARs were predominantly visualized by the CAM2 reagent in cultured hippocampal neurons. Data points mean ± SEM. (f) After chemical labeling by 1 µM of CAM2(Ax488), the neurons were incubated for 24 h at 37 °C to promote internalization of labeled AMPARs and subjected to TB treatment. Orange square ROIs indicated in upper panels are expanded in lower panels. Scale bars, 10 µm in upper panels and 5 µm in lower panels.
(g) Comparison of I TB /I initial obtained from hippocampal neurons after incubation at 37 °C for 0 h (in d, n = 12), 12 h (the image not shown, n = 11) or 24 h (in f, n = 12).
This data also supports that surface-exposed AMPARs are predominantly visualized immediately after CAM2 labeling. Extracellular pH was stepwise changed from 7.5 to 5.5, to 6.0, to 6.5, to 7.0, to 7.5 and finally to 8.0. The cellular fluorescence at different pH was monitored (n = 41).

Synthesis of compound 18
A solution of 17 (32 mg, 69 µmol) in 30 % fuming H 2 SO 4 (1 mL) was stirred for 12 h on ice. Dry dioxane (5 mL) and Et 2 O (10 mL) were poured to the solution and the resulting supernatant was removed completely and the residue was dried in vacuo. The  atmosphere. After removal of the solvent by evaporation, the residue was precipitated by CH 2 Cl 2 (2 mL) and Et 2 O (3 mL) to give 19 (10 mg) containing some impurities, which was used for the next step without further purification.

Synthesis of compound 21
A solution of crude 20 (10 mg) and 0.5 N LiOH (130 µl, 66 µmol) in MeOH (1 mL) was stirred for 12 hr at r.t. After removal of solvent, DCM (1 mL), and TFA (1 mL) were added and stirred for 12 hr at r.t. After removal of the solvent by evaporation, the residual TFA was further removed azeotropically with toluene (x2) to give crude 21.
The compound was used for the next step without further purification.