Johnson Pilot Project Summary
The goal of this application is to develop an easily fabricated probe with an array of nanoelectrodes for the measurement of dopamine (DA) and other electroactive molecules in living brain tissue. DA encodes reward signals and exerts control over movement by its release in the nucleus accumbens and dorsal striatum. The traditional view of DA release is that diffuses from sites of release to distant locations hundreds of nanometers to several millimeters away, thereby causing widespread receptor activation on many target cells, a phenomenon known as volume transmission. However, recent studies suggest that certain DA-related functions necessitate a higher degree of spatiotemporal precision, with signaling relying on rapid release and subsequent diffusion to receptors on the micron scale. These factors are important when considering the underlying mechanisms of diseases such as Parkinson’s disease (PD), a progressive neurodegenerative disorder characterized by the death of dopaminergic neurons in the substantia nigra parts compacta and decreased DA release in the striatum.
Fast-scan cyclic voltammetry (FSCV) is a method of choice for measuring DA release and uptake in brain tissue on physiologically relevant concentrations and timescales. To obtain FSCV recordings, researchers commonly use single carbon-fiber microelectrodes, which are typically on the order of five to ten microns in diameter and tens to hundreds of microns long, making spatiotemporally resolved studies of DA signaling difficult. To obtain DA release measurements that are spatiotemporally resolved, we will leverage the outstanding expertise and facilities at the University of Kansas Nanofabrication Core Facility to fabricate and characterize nanoarray probes and use them to evaluate dopamine release and uptake properties in live, whole brains from zebrafish treated with rotenone, a mitochondrial complex I inhibitor. Treatment with this chemical re-capitulates many of the pathophysiological features of PD. We will electrically evoke DA release by stimulating cell bodies in the posterior tubercle (PT) and measuring dopamine release at the projections in the dorsal nucleus of the ventral telencephalon (Vd). This pathway is analogous to dopaminergic pathways involved with the control of movement and learning in rodents. We will also label dopamine transporter molecules in zebrafish whole brains with fluorescent probes synthesized by the Synthetic Chemical Biology Core, and image them with two-photon excitation (2PE) microscopy. This orthogonal method will allow us to compare our functional measurements of DA release with measurements of dopaminergic innervation.
Our specific aims are to: 1) Quantify stimulated DA release in the Vd of PD model and control zebrafish and 2) Determine relative densities dopaminergic terminals in PD model and control zebrafish. This work will provide previously unattainable insights into dopaminergic signaling in health and disease, as well as provide a new tool that can be used to measure multiple neurotransmitters simultaneously.