Microscopic Visualization of Metabotropic Glutamate Receptors on the Surface of Living Cells Using Bifunctional Magnetic Resonance Imaging Probes

Abstract

A series of bimodal metabotropic glutamate-receptor targeted MRI contrast agents has been developed and evaluated, based on established competitive metabotropic Glu receptor subtype 5 (mGluR 5 ) antagonists. In order to directly visualize mGluR 5 binding of these agents on the surface of live astrocytes, variations in the core structure were made. A set of gadolinium conjugates containing either a cyanine dye or a fluorescein moiety was accordingly prepared, to allow visualization by optical microscopy in cellulo. In each case, surface receptor binding was compromised and cell internalization observed. Another approach, examining the location of a terbium analogue via sensitized emission, also exhibited nonspecific cell uptake in neuronal cell line models. Finally, biotin derivatives of two lead compounds were prepared, and the specificity of binding to the mGluR 5 cell surface receptors was demonstrated with the aid of their fluorescently labeled avidin conjugates, using both total internal reflection fluorescence (TIRF) and confocal microscopy.

Electrochemical Modulation of the Fluorescence of Cyanine Dye Cy5

“…Organic fluorescent dyes have been widely used in spectral sensitization, color photography or solar cells 1–3. In the last few decades, particularly, organic dyes as powerful fluorophores, have been commonly used in biological research field 4, 5. Since the discovery of their light‐induced photoswitching capability, extensive studies have been conducted in terms of applications of organic dyes in single molecule localization microscopy (SMLM) 6–8.…”

Magnetic resonance and optical imaging probes for NMDA receptors on the cell surface of neurons: synthesis and evaluation in cellulo

“…[7][8][9]13 The other compounds, ligands and Gd complexes not reported below are described in the ESI. † Ligand and complex synthesis (3-Amino-2-hydroxypropyl)carbamic acid 1,1-dimethylethyl ester, 11b 1.…”

“…This rationale has driven the recent development of contrast agents that selectively target the various glutamate receptors that are found on the cell surface of astrocytes in high abundance. [7][8][9] Such responsive contrast agents must be able to bind selectively and reversibly to the receptor, so that changes in local contrast agent levels can be modulated following the glutamate bursts that periodically occur following a stimulus. Given that release of glutamate from a pre-synaptic cell takes place over a period of milliseconds and should displace the contrast agent from the cell surface receptor, MRI may be able to monitor the restoration to equilibrium that occurs over a period of a second or two.…”

Molecular fMRI

“…The resulting complexes displayed millimolar affinity for glutamate, GABA, and glycine, and neurotransmitter binding was accompanied by r 1 changes of up to 81% in buffer. Another T 1 agent synthesized by Mishra et al (2013) was designed to sense glutamate by a mechanism involving competitive binding to mGluR 5 receptors, somewhat like receptor displacement approaches used for PET (Laruelle, 2000). This probe bound to mGluR 5 -expressing cells in culture and produced MRIdetectable T 1 changes at micromolar concentrations in vitro.…”

“…Cytosolic Ca 2ϩ fluctuations extend from basal concentrations near 0.1 M to levels as high as 10 M near synapses and are already so heavily buffered in cells that addition of calcium indicators produces minimal perturbation of calciummediated signaling processes (Meldolesi, 2002). Powerful small molecule fluorescent sensors for calcium have been available since the 1980s (Grynkiewicz et al, 1985;Tsien et al, 1985); and after many successive stages of refinement, genetically encoded GFP-based calcium indicators descended from those introduced in the late 1990s are now also in wide use (Miyawaki et al, 1997;Nagai et al, 2001;Rose et al, 2014). Given this precedent, the development of calcium-dependent molecular fMRI methods is an obvious priority.…”