Real-Time Monitoring of Striatal GPCR Mediated cAMP Signaling Using Genetically Encoded Fluorescent Sensors

Striatal neuromodulation through G-protein-coupled receptors (GPCRs) regulates complex voluntary motor actions, involving decision-making, learning, and action selection. The dorsal striatum integrates information from neuromodulatory inputs from the thalamus, midbrain, and various parts of cortex, as well as intrinsic neuromodulators within in its microcircuity. The integration of these signals initiates the communication of information through the basal ganglia to control action learning and performance. Many of these inputs form synapses onto indirect and direct-pathway projection medium spiny neurons (MSN). Both subpopulation of MSNs contains GPCRs that can oppositely regulate the cAMP-PKA signaling pathway. This signaling pathway is critical in the modulation of synaptic transmission, long-term synaptic plasticity and learning and memory. In the striatum, the dynamic interplay between dopamine and cAMP-PKA signaling is important for the induction of long-term depression. However, the spatial and temporal dynamics of these intracellular effector molecules in real-time during the induction of synaptic plasticity and behavior remain unclear. Using newly optimized intensity- and FRET-based genetically encoded cAMP biosensors together with optical techniques, we can now measure activity-induced changes in cAMP accumulation in real-time in live brain slices and in freely moving animals. Simultaneous recordings of activity-induced cAMP transients detected by cAMP Difference Detector in situ (cADDis) intensity-based sensor and dopamine transients were performed using slice photometry and fast-scan cyclic voltammetry, respectively. Single pulse electrical stimulation generates increased cAMP in D1 dopamine receptor-expressing MSNs and decreased cAMP in D2 receptor expressing MSNs. These signaling changes coincide with increased extracellular dopamine. We are currently examining the kinetics of the dopamine and cAMP signals. Both transients can be altered by changes in stimulation duration and frequency, and are blocked by tetrodotoxin, indicating that they are driven by neuronal activation. Our findings indicate the feasibility of using optical biosensors combined with traditional methods to investigate precise temporal dopamine-cAMP dynamics in real-time in the striatum.

Learning Objectives:

1. Learn about newly developed and optimized genetically encoded fluorescent sensors to measure second messenger effector molecules

2. Explain optical imaging tools to measure these sensors in vitro (live brain slices) and in vivo during freely moving behaviors