The neurotransmitter dopamine is pivotal in decision making, habit and motor learning as well as in drug abuse, schizophrenia, attention deficit hyperactive disorder (ADHD), and Parkinson's disease. This joint effort from three NIH-funded laboratories explores fundamental mechanisms of both pre- and post-synaptic dopaminergic neurotransmission using novel tools. The team includes a chemistry laboratory (Sames) that designs and produces the optical probes, a neurophysiology laboratory that characterizes and analyzes the synaptic release of dopamine using optical recordings at synaptic level (Sulzer), and a molecular neuroscience laboratory that uses a variety of optical approaches to elucidate the structure, activation, and pharmacology of dopamine receptors (Javitch). The three groups have been collaborating intensively for the past five years, and this application results from these collaborations. The novel approaches described here use the first optical methods developed to directly visualize neurotransmitter release, using optical probes known as fluorescent false neurotransmitters (FFNs). The team has now developed a first generation of ratiometric neurotransmitter probes that provide a means to measure pH in synaptic vesicles within neuronal terminals. The probes have also been adapted to simultaneously measure neurotransmission and synaptic vesicle fusion at individual synaptic terminals. At the postsynaptic side, additional probes promise to measure receptor occupancy at unprecedented temporal resolution. While our current data demonstrate the utility of this new generation of optical probes using multiphoton imaging in the acute brain slice, the millisecond-scale events involved in neurotransmission and receptor and transporter activation require virtually simultaneous excitation at two wavelengths, thus necessitating two light sources. We therefore outline how a multiphoton laser will enable multiple and important enhancements to the research of this multidisciplinary group of research teams.
Aim 1 outlines how synaptic vesicle fusion and neurotransmitter release will be recorded simultaneously Aim 2 outlines how synaptic vesicle pH will be determined in living synaptic terminals.
Aim 3 outlines how caged dopamine used with fluorescence energy transfer will determine the structure, function, and pharmacology of D2 dopamine receptors at unprecedented kinetic resolution.
|Tang, Guomei; Gudsnuk, Kathryn; Kuo, Sheng-Han et al. (2014) Loss of mTOR-dependent macroautophagy causes autistic-like synaptic pruning deficits. Neuron 83:1131-43|
|CebriÃ¡n, Carolina; Zucca, Fabio A; Mauri, Pierluigi et al. (2014) MHC-I expression renders catecholaminergic neurons susceptible to T-cell-mediated degeneration. Nat Commun 5:3633|
|Hernandez, Daniela; Torres, Ciara A; Setlik, Wanda et al. (2012) Regulation of presynaptic neurotransmission by macroautophagy. Neuron 74:277-84|