No abstract

Functional MRI has become an important tool of researchers and clinicians who seek to understand patterns of neuronal activation that accompany sensory and cognitive processes. However, the interpretation of fMRI images rests on assumptions about the relationship between neuronal firing and hemodynamic response that are not firmly grounded in rigorous theory or experimental evidence. Further, the blood-oxygen-level-dependent effect, which correlates an MRI observable to neuronal firing, evolves over a period that is 2 orders of magnitude longer than the underlying processes that are thought to cause it. Here, we instead demonstrate experiments to directly image oscillating currents by MRI. The approach rests on a resonant interaction between an applied rf field and an oscillating magnetic field in the sample and, as such, permits quantitative, frequency-selective measurements of current density without spatial or temporal cancellation. We apply this method in a current loop phantom, mapping its magnetic field and achieving a detection sensitivity near the threshold required for the detection of neuronal currents. Because the contrast mechanism is under spectroscopic control, we are able to demonstrate how ramped and phase-modulated spin-lock radiation can enhance the sensitivity and robustness of the experiment. We further demonstrate the combination of these methods with remote detection, a technique in which the encoding and detection of an MRI experiment are separated by sample flow or translation. We illustrate that remotely detected MRI permits the measurement of currents in small volumes of flowing water with high sensitivity and spatial resolution. current imaging | EEG | magnetoencephalography I n some cases, including living neural tissue, electric charges moving within the sample produce oscillating magnetic fields that can be visualized by MRI methods. The imaging of current distributions by MRI has developed significantly over the last 20 years, with early applications being directed toward the imaging of current density and conductivity in model systems (1, 2) and later in vivo (3-5). However, the primary focus in the development of current imaging is the possibility of directly imaging neuronal currents.While the currents generated by a single neuron are far too small to measure, detectable magnetic field changes on the order of 0.1-1 nT (6) may result from synchronized postsynaptic currents in a large number of neurons. The frequency of oscillatory neural activity is also extremely significant. In addition to the previously demonstrated importance of alpha wave (∼10 Hz) processes (7), a body of recent work has identified the importance of brain activity in the gamma and high gamma frequency ranges (25-250 Hz) (8-10) to the synchronization of anatomically distant centers. To date, most successful approaches to the mapping of these frequencies have involved the implantation of electrodes in direct contact with the brain, usually during a surgical procedure. A noninvasive measurement of oscillating c...

Molecular wires comprising a Ru(II)-or Re(I)-complex head group, an aromatic tail group, and an alkane linker reversibly inhibit the activity of the copper amine oxidase from Arthrobacter globiformis (AGAO), with K i values between 6 M and 37 nM. In the crystal structure of a Ru(II)-wire:AGAO conjugate, the wire occupies the AGAO active-site substrate access channel, the trihydroxyphenylalanine quinone cofactor is ordered in the ''off-Cu'' position with its reactive carbonyl oriented toward the inhibitor, and the ''gate'' residue, Tyr-296, is in the ''open'' position. Head groups, tail-group substituents, and linker lengths all influence wire-binding interactions with the enzyme.diimine ͉ topaquinone ͉ metalloenzyme ͉ active site C opper and quinone containing amine oxidases (EC 1.4.3.6) catalyze the oxidative deamination of primary amines to the corresponding aldehydes with concomitant generation of ammonia and hydrogen peroxide.Each subunit of these homodimeric enzymes contains a deeply buried active site comprised of a single type II (''non-blue,'' square-pyramidal) copper atom and an organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (topaquinone or TPQ) (1, 2). The finding that the human vascular adhesion protein (HVAP-1) is a copper amine oxidase (CuAO) has heightened interest in the mechanism and inhibition of these enzymes (3). With the potential for therapeutic applications, research has focused on elucidation of the factors that govern inhibitor sensitivity and selectivity.We are exploring the potential of channel-blocking metaldiimine wire complexes to function as highly selective inhibitors of CuAOs. We chose phenylethylamine oxidase from Arthrobacter globiformis for initial study, owing to its ease of expression and purification as a C-terminal Strep-tag II fusion protein (4). Our choice of metal-diimine wires was based on the results of extensive investigations of their conjugates with cytochrome P450cam, which have revealed structural features of conformational states that likely are involved in steps of the catalytic cycle of the enzyme (5-9). Similar molecular wires have been used in attempts to measure the reduction potentials of deeply buried protein cofactors; indeed, in experiments of relevance here, a diethylaniline-tipped triphenylene wire coupled to a gold electrode allowed electrochemical characterization of Arthrobacter globiformis amine oxidase (AGAO) cofactor TPQ (10). Binding of the wire in the active-site channel was not established independently but could be inferred from the efficiency of electron tunneling from the electrode to the buried cofactor.We have designed and synthesized a series of highly potent channel-blocking inhibitors of AGAO. The crystal structure of a Ru-wire:AGAO conjugate clearly demonstrates that the wire resides in the active-site channel; it also reveals key aspects of active-site topology and conformational mobility. Furthermore, variations in binding in response to changes in wire sensitizer, substrate, and linker compositions have led to particula...