Towards functional NMDA-receptor MR-imaging: Novel probes based on competitive antagonists

For neuroimaging techniques such as positron emission tomography and optical imaging brain receptor-targeting is a widely used concept, either using probes based on receptor antagonists or agonist. So far, applying competitive binding approaches for brain functional magnetic resonance imaging (fMRI) has not been demonstrated. Our idea was therefore to develop responsive MRI contrast agents (CAs) based on competitive antagonists to the N-methyl-D-aspartate (NMDA) receptor, an ionotropic glutamate receptor that plays an important role in controlling synaptic plasticity and memory function.[1] Ultimately, such CAs would allow to monitor glutamate activity at NMDA receptors (NMDARs) and thus neuronal activity itself. For such a CA to be responsive, release of glutamate from the pre-synapse will detach the CA from the receptor, in turn leading to a reduction in image contrast, followed by a restoration of equilibrium and again binding of the CA to the receptor. It has been shown, that these events happen over a period of a few seconds allowing data acquisition with modern fast MR-techniques.[2] Here we report the in vitro evaluation of a series of NMDAR targeted CAs, that are based on established competitive NMDAR-antagonists[3] coupled to DOTA-derived gadolinium chelates (Fig. 1A). Cellular labeling, cytotoxicity, receptor binding and reversibility were studied on the NMDAR-expressing (shown by immunofluorescence) neuronal cell line model NSC-34. Binding affinity in cultured cells (Fig. 1B) showed that two of the compounds (Gd.L2 and Gd.L4) increased the cellular relaxation rate R1,cell up to 170�11 and 176�4 of control, respectively (200 �M, 45min., 37�C). MRI-measurements were done on a 3T human whole body scanner. No cytotoxic effects with the concentrations used (24h incubation) were seen. Receptor binding and reversibility were demonstrated using a modified version of Gd.L4, with a trans-substituted biotin moiety appended to the macrocyclic core (Gd.L5, Fig. 1C). Cell-surface binding was visualized after adding an AvidinAlexaFluor� 488 conjugate, which binds with high specificity to the biotin moiety, while the receptor-binding moiety of the CA binds to the receptor on the cell membrane. Live cell confocal microscopy showed labeling of the cell membrane by Gd.L5 (green) in a pit-like manner and co-localization with a cell membrane specific marker (red, Figure 1D). Furthermore, a glutamate wash (1 mM) demonstrated that Gd.L5 is removed from the cell surface (Fig. 1E). In conclusion, we were able to identify two promising novel NMDAR-targeted MRI contrast agents. These CAs are based on the structures of competitive antagonists to NMDAR and we were able to demonstrate specific receptor binding and reversibility, which is essential for functional measurements of receptor-activity. Thus, our results indicate the possibility of using MRI for brain functional measurements based on competitive binding approaches