Calcium Sensors for Molecular FMRI

The development of minimally invasive direct readouts of neural activity is one of the greatest challenges facing neuroscience today. Our recent work has shown that it is possible to perform high resolution functional magnetic resonance imaging (fMRI) of molecular-level phenomena using MRI contrast agents sensitive to hallmarks of neurotransmitter release. An even more valuable contribution would be the creation of calcium sensors suitable for molecular fMRI of intracellular neural signaling processes. Functional imaging performed with these sensors would combine the noninvasiveness and whole-brain coverage of MRI with the molecular specificity and broad applicability of established optical calcium neuroimaging techniques. Calcium-dependent fMRI will be a breakthrough technique for analysis of neural circuits in animals, with potential longer term applications in humans. The technique could achieve cellular resolution in conjunction with ultrahigh field MRI scanners and cell labeling techniques. A major hurdle in realizing this advance is the creation of effective calcium-dependent MRI contrast agents, however. This proposal describes strategies for creating novel MRI calcium probes suitable for molecular fMRI, as well as initial experiments that validate the approach in animals. Innovations include the rational design of membrane permeable probes themselves as well as approaches for in vivo calcium imaging and genetically targeted applications. In Aim 1, we synthesize MRI calcium sensors based on paramagnetic cell-permeable aromatic chelates and characterize them in vitro. In Aim 2, we form acetomethoxy derivatives of the calcium probes and validate them first in cell culture and then in rats, using a somatosensory stimulation paradigm. Results of Aim 2 will direct further refinement of the probes, if necessary. In Aim 3, we adapt the calcium sensors for intracellular trapping by selective esterases that will promote probe accumulation in genetically targeted cells. This technique will provide a means for cell type-specific and in some cases individual cell-specific functional imaging of dynamic calcium levels in the brain.