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Responsive MR Imaging Probes to Monitor Synaptic Glutamate Fluctuations in the Brain

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http://pubman.mpdl.mpg.de/cone/persons/resource/persons83941

Gottschalk,  S
Department High-Field Magnetic Resonance, Max Planck Institute for Biological Cybernetics, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons84084

Mishra,  A
Department Physiology of Cognitive Processes, Max Planck Institute for Biological Cybernetics, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons83903

Engelmann,  J
Department High-Field Magnetic Resonance, Max Planck Institute for Biological Cybernetics, Max Planck Society;

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Gottschalk, S., Mishra, A., Engelmann, J., & Parker, D. (2011). Responsive MR Imaging Probes to Monitor Synaptic Glutamate Fluctuations in the Brain. Talk presented at 6th European Molecular Imaging Meeting (EMIM 2011). Leiden, Netherlands.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0013-BB96-B
Abstract
mediator of excitatory signals in the nervous system and is involved in nearly all aspects of normal brain functioning (cognition, memory, learning). Our idea was to develop glutamate ‘responsive’ magnetic resonance imaging (MRI) contrast agents (CAs) to image changes in specific brain regions upon neural activation. As CAs directly responsive to glutamate would not be feasible due to the very short half-life of glutamate in the extracellular space, we chose CAs that bind to glutamate receptors instead (to be specific metabotropic glutamate-receptor subtype 5 (mGluR5)), by this increasing image contrast. Ideally, upon glutamate-binding to the receptor (e.g. after glutamaterelease at the synapse) the CA will be released, hence leading to a reduction in image-contrast, followed by a restoration of equilibrium and re-binding of the CA to the receptor. These events are believed to occur over a period of seconds allowing data acquisition using modern FLASH pulse techniques[1]. Here, we present a proof-of-concept study for such ‘indirect’ glutamate-responsive MRI CAs. Methods: We have designed and synthesized different prospective CAs derived from various potent mGluR5-receptor antagonists (alkynes like MPEP, MTEP and dipyridyl/heterobiaryl amides) coupled to DOTA-derived macrocyclic lanthanidechelates. The CAs were evaluated in cultured primary cortical rat astrocytes, expressing mGluR5 (verified by immunofluorescence). MRI-measurements to examine the ability of the CAs for cellular labeling were done with a 3T human whole body scanner. Antagonistic potency of the CAs was assessed with a calcium fluorescence assay, by which glutamate induced intracellular calcium-transients mediated by mGluR5 were measured. Antagonistic activity of the CAs was calculated as changes in EC50 of glutamate. Receptor binding was measured for the dipyridyl derivaties, as these compounds have an inherent fluorescence that changes upon binding. Commercially available receptor membrane preparations containing human mGluR5A were used for these experiments. Results: Two of the gadolinium complexes retained significant antagonistic activity, one in each structural class. For the alkyne-derivative, about a threefold increase of the EC50(glutamate) (100μM CA, 15min, P<0.001) was found while under similar conditions the cellular relaxation rate R1,cell increased to 126 of control (100μM, 45 minutes incubation time, P<0.001). The CA derived from dipyridyl amides increased the EC50(glutamate) about fourfold (p<0.001) and the R1,cell to 115 (p<0.05). Fluorescence measurements of the latter CA showed enhanced emission upon binding to mGluR5-membrane preparations. This was reversed when increasing concentrations of glutamate were added, consistent with the a reversibility of CA-receptor binding Conclusions: Using primary rat astrocytes as cellular model system to investigate newly developed glutamate-responsive MRI contrast agents, we were able to identify two promising candidates. These CAs are based on the structures of antagonists to mGluR5 and our studies establish the validity of the concept, by which it might be possible to use MRI to image transient changes in the neurotransmitter glutamate.