We propose the application of artificial neurotransmitter receptors (ANR) for the monitoring of dopamine levels in neuronal tissues as well as in prefrontal cortex and basal nuclei of intact mice. In the first step, existing and newly prepared fluorescent ANRs will be tested under the conditions of 2-photon microscopy and single photon microendoscopic imaging. Parameters that will be changed are the zeolite and dye identity, dye loading, surface functionalization and the size of the zeolites. Kinetic rates of signal appearance, which is related to the neurotransmitter uptake rates, will be measured. Additional ANRs will be prepared that contain widely used 2-photon absorbing dyes such as DAPI, either directly as the “indicator dye” or as the “antenna dye” (in combination with FRET to the indicator dye). The sensing performance of such ANRs for different aromatic amine neurotransmitters will be evaluated in buffers and biological media (e.g. cell culture media). Promising ANR candidates will be then tested first by application to acute brain slices using single-photon and two-photon excitation. To image dopamine signalling in the intact brain, ANRs will be directly delivered to the brain region of interest by stereotaxic injection.
Regions of the prefrontal cortex accessible to two-photon microscopy will be examined with in vivo two-photon microscopy. Deep regions such as the striatum or nucleus accumbens will be imaged via implanted microendoscopes using either two-photon imaging in head-fixed mice or single-photon imaging with miniature microscopes in freely behaving mice. This approach promises systemic readouts of dopamine action in the context of the behaving animal, allowing to directly link neuronal and circuit mechanisms related to dopamine signalling with distinct behaviours.
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