(In questions of science, the authority of a thousand is not worth the humble reasoning of a single individual.*)
In vivo imaging of neurotransmitter volume transmission
The volume conduction of neurotransmitters plays a fundamental role in neuromodulation. This includes small molecule transmitters, which are key players in mental disorders and addiction, and peptide neurotransmitters, which are important for the neuroendocrine system and the control of vascular tone and blood flow. Yet there are currently limited means to detect neurotransmitters in vivo.
To address this deficit we devised cell-based neurotransmitter fluorescent engineered reporters, which we refer to as CNiFERs. A CNiFER is a clonal cell-line that is engineered to express two proteins: (i) a specific G-protein coupled receptor that is of the Gq family or transformed to be of that family; and (ii) a genetically encoded FRET-based Ca2+ sensor that detects changes in intracellular [Ca2+]. Binding to the metabotropic Gq-coupled receptor under physiological conditions leads to an elevation in cytosolic [Ca2+] and thus a change in fluorescence that provides a direct and rapid readout of local neurotransmitter activity.
CNIFERs are implanted into rodent cortex and imaged with two-photon laser scanning microscopy to measure neurotransmitter concentration in the awake and behaving animal. They have one to ten nanoMolar sensitivity, about a one second response time for the onset of a change in transmitter concentration, and a differential resolution of about five seconds. We acheive a spatial resolution down to 50 micrometers.
Ongoing projects are in collaboration with the Slesinger laboratory (MSSM). We currently address: (1) the role of peptide neuromodulators modulators in the control of blood flow in cortex; (2) the volitional control of neuromodulators; and (3) the temporal dynamics of aceytlcholine and other neuromodulators during the transition between sleep and wakefulness. Planned work involves the use of CNiFERs in subcortical areas in conjunction with fiber optics.