Probing the brain with molecular fMRI

Abstract

One of the greatest challenges of modern neuroscience is to incorporate our growing knowledge of molecular and cellular-scale physiology into integrated, organismic-scale models of brain function in behavior and cognition. Molecular-level functional magnetic resonance imaging (molecular fMRI) is a new technology that can help bridge these scales by mapping defined microscopic phenomena over large, optically inaccessible regions of the living brain. In this review, we explain how MRI-detectable imaging probes can be used to sensitize noninvasive imaging to mechanistically significant components of neural processing. We discuss how a combination of innovative probe design, advanced imaging methods, and strategies for brain delivery can make molecular fMRI an increasingly successful approach for spatiotemporally resolved studies of diverse neural phenomena, perhaps eventually in people.

Figure 1

Calcium sensors potentially suitable for molecular fMRI. (a) The 5,5'-difluoro-BAPTA imaging agent used in the approach of ref. [10]. Calcium binding (blue) causes reversible changes in the electronic structure of the agent, and shift the spectroscopic signals associated with the fluorine atoms (magenta). (b) Standard proton MRI scan (left), fluorine MRI scan (middle), and fluorine MRI with CEST contrast (right) showing fluorinated BAPTA in the presence of calcium, magnesium, or zinc ions, as indicated in the left panel. Only calcium induces substantial signal change in the CEST image, allowing specific detection of calcium using the agent. (c) A gadolinium-based calcium sensor described in ref. [16]. In the presumed mechanism of this probe, one or more of the ligands that coordinate Gd³⁺ (green) in the calcium-free form (left) is sequestered upon calcium binding (right), freeing up additional sites for water molecules (magenta) to interact with Gd³⁺ and induce T₁ MRI contrast enhancement. Conjugation of the agent to a nanoparticle scaffold (gray, not to scale) increases retention time of the agent in tissue. (d) MRI contrast change following injection of the probe into mouse kidney, before (left) vs. after (right) stimulation with intravenous CaCl₂. (e) Mechanism of calcium-responsive magnetic nanoparticle sensors in ref. [18]. Clustering of the nanoparticles (gray) is driven by polydendate interactions between calmodulin (red) and a calmodulin-binding peptide (green) attached to a tetrameric molecular scaffold (magenta). (f) Reversible clustering and declustering is measured within seconds of exposure of the sensors to excess Ca²⁺ or EDTA. Inset shows substantial T₂-weighted MRI signal differences measured between EDTA (black arrowhead) and calcium-enriched (blue arrowhead) conditions for nonfunctional control nanoparticles (left) or functional bicomponent sensors (right).

Figure 2

Hemodynamic fMRI of neural activity signatures resolved at high spatiotemporal resolution. (a) Yu et al. [24] expressed channelrhodopsin-2 (ChR2) in rat somatosensory cortex and evoked neural responses using optogenetic stimulation via an implanted fiber (blue). Top panel shows orientation of MRI scans in (b–d) and bottom panel shows ChR2 expression pattern in a perpendicular slice. (b) A technique called multi-gradient imaging was used to identify single vessels based on contrast signatures in high resolution MRI scans (field of view corresponds to yellow slices in (a)). (c) Features in two forms of hemodynamic fMRI—blood oxygen level-dependent contrast (BOLD, top right) and cerebral blood volume-dependent contrast (CBV, bottom right)—could be correlated with regions identified as venules (top left) or arterioles (bottom left) based on data as in (b) (green boxed region). Figure adapted from ref. [24].

Figure 3

Molecular fMRI using polypeptide-based imaging agents. (a) Structure of a P450-BM3h variant engineered by Brustad et al. [83] for selective sensing of the neurotransmitter dopamine in T₁-weighted MRI. The sensing mechanism arises from the ability of dopamine (DA, blue) binding to regulate water access to the paramagnetic heme group (green label) in the core of the protein. (b) Dopamine binding to the P450 derivative alters the strength (relaxivity) of the contrast agent, as shown by in vitro measurements in PBS and dopamine-containing buffers; inset shows corresponding MRI images. (c) Quantitative mapping of dopamine release in rat ventral striatum, elicited by MFB stimulation in the experiments of Lee et al. [31]. Panels depict anatomical MRI (grayscale) and atlas divisions (white) in three striatal slices of 1 mm thickness, with color overlay indicating peak dopamine release concentrations determined from raw MRI signal time courses. Scale bar = 2 mm; adapted from ref. [31]. (d) Strategy of hijacking hemodynamic responses using molecular agents. Vasoprobes such as the potent vasodilator CGRP agonize receptors on vascular smooth muscle cells (left), promoting relaxation and vasodilation (middle), and leading to changes in blood flow that give rise to MRI signals detectable by hemodynamic molecular fMRI, as well as other imaging techniques. (e) Application of vasoprobe-based molecular imaging for detecting enzyme activity. The schematic (top) shows that a caged vasoprobe is inhibited by a blocking domain; in the presence of an enzyme (red), the blocking domain is removed by cleavage of recognition sequence connecting the two (red line segment). The example at bottom demonstrates this principle applied to detection of the protease caspase-3. Enzyme cleaved (right) but not uncleaved (left) probe produces engineered hemodynamic responses upon injection into rat brain, permitting visualization by hemodynamic MRI. Scale indicates statistical significance of the detection. (f) Vasoprobes can function as gene reporters by being expressed and secreted from genetically engineered cells (top). Engineered CGRP-expressing human embryonic kidney cells were implanted into rat brain and could be detected by T₂-weighted MRI after 24 hours (bottom left). MRI signal changes coincided with co-expression of a fluorescent protein (mKate), visualized by postmortem histology (bottom right). Control cells not expressing the vasoprobe did not produce substantial MRI signatures. Scale bar = 2 mm; (d–f) adapted from ref. [55].

Figure 4

Delivery of imaging probes across the blood-brain barrier (BBB). (a) Simplified schematic demonstrating the opening of the tight junctions between the endothelial cells of the BBB upon application of ultrasound waves in conjunction with intravascular microbubbles (bubbles not shown). Penetration of multiple blood-born species (colored shapes) following BBB disruption takes place, without specificity for imaging reporters per se. (b) Kovacs et al. [64] demonstrated the use of an implantable ultrasound transducer to transiently open the BBB of cancer patients in a clinical trial. (c) A gadolinium-based MRI contrast agent was intravenously administered to monitor BBB opening; leakage of the contrast agent into the brain into the sonicated region (yellow box) can be discerned by comparing images acquired before (left) vs. after (right) sonication. Figure adapted from ref. . (d) Mechanisms of spontaneous BBB penetration by imaging agents: (left) transcellular diffusion by lipophilic molecules (red); (middle) carrier-mediated transport (CMT) of imaging agents (star) attached to CMT substrates (black diamond); and (right) and receptor-mediated transport (RMT) of receptor-targeted complexes (light purple circle) incorporating an imaging agent (star), which in some cases may be released once the complex enters the brain. (e) Diagram of a typical Trojan horse construct based on an antibody with variable domains targeted against human insulin receptor (purple), conjugated to imaging agents (stars). (f) Boado et al. [71] demonstrated Trojan horse-mediated brain delivery of a radiolabeled therapeutic anti-HIR conjugate in nonhuman primates. Autoradiography of postmortem brain sections from a monkey treated with unconjugated agent (left) vs. a monkey treated with anti-HIR-conjugated probe (right) illustrate strong brain uptake dependent on the anti-HIR construct. Radiolabel density indicated by yellow-red-back color scale. This technique could be adapted for delivery of MRI contrast agents, although only the most potent agents are likely to be appropriate. Figure adapted from ref. .