Investigation of a Calcium-Responsive Contrast Agent in Cellular Model Systems: Feasibility for Use as a Smart Molecular Probe in Functional MRI

Responsive or smart contrast agents (SCAs) represent a promising direction for development of novel functional MRI (fMRI) methods for the eventual noninvasive assessment of brain function. In particular, SCAs that respond to Ca" may allow tracking neuronal activity independent of brain vasculature, thus avoiding the characteristic limitations of current fMRI techniques. Here we report an in vitro proof-ofprinciple study with a Ca"-sensitive, Gd"-based SCA in an attempt to validate its potential use as a functional in vivo marker. First, we quantified its relaxometric response in a complex 3D cell culture model. Subsequently, we examined potential changes in the functionality of primary glial cells following administration of this SCA. Monitoring intracellular Ca" showed that, despite a reduction in the Ca" level, transport of Ca' through the plasma membrane remained unaffected, while stimulation with ATP induced Ca"-transients suggested normal cellular signaling in the presence of low millimolar SCA concentrations. SCAs merely lowered the intracellular Ca' level. Finally, we estimated the longitudinal relaxation times (T1) for an idealized in vivo fivIRI experiment with SCA, for extracellular Ca' concentration level changes expected during intense neuronal activity which takes place upon repetitive stimulation. The values we obtained indicate changes in T1 of around 1-6%, sufficient to be robustly detectable using modern MRI methods in high field scanners. Our results encourage further attempts to develop even more potent SCAs and appropriate fMRI protocols. This would result in novel methods that allow monitoring of essential physiological processes at the cellular and molecular level.