Summary Reinforcement learning models postulate that dopamine (DA) releasing neurons (DANs) encode information about action and action outcome and provide a teaching signal to striatal spiny projection neurons (SPNs) in the form of DA release 1 . DA is thought to guide learning via dynamic and differential modulation of protein kinase A (PKA) in each class of SPN 2 . However, the real-time relationship between DA and SPN PKA remains untested in behaving animals. Here, we monitor the activity of DANs, extracellular DA levels, and net PKA activity in SPNs in the nucleus accumbens in mice during learning. We find positive and negative modulation of DA that evolves across training and is both necessary and sufficient to explain concurrent fluctuations in SPN PKA activity. The modulations of PKA in SPNs that express type-1 and type-2 DA receptors are dichotomous such that they are selectively sensitive to increases and decreases in DA, respectively, which occur at different phases of learning. Thus, PKA-dependent pathways in each class of SPNs are asynchronously engaged by positive or negative DA signals during learning.

All cells respond to extracellular signals by altering their intracellular biochemical state. In neurons, such signaling regulates many aspects of cell and synapse biology and induces changes that are thought to be important for nervous system development, its adaptation in the face of a changing environment, and ongoing homeostatic maintenance. Although great advances have been made in developing novel fluorescent reporters of intracellular signaling as well as in methods of fluorescence detection for use in freely moving animals, these approaches have generally not been combined. Thus, we know relatively little about how the intracellular biochemical state of neurons, and other cell classes, is dynamically regulated during animals’ behavior. Here we describe a single multi-mode fiber based f luorescence li fetime p hotometry system (FLiP) designed to monitor the state of fluorescence reporters of biochemical state in freely moving animals. We demonstrate the utility of FLiP by monitoring the lifetime of FLIM-AKAR, a genetically encoded fluorescent reporter of PKA phosphorylation, in populations of direct and indirect pathway striatal projection neurons in mice receiving food rewards. We find that the activity of PKA in each pathway is transiently regulated by reward acquisition, with PKA phosphorylation being enhanced and repressed in direct and indirect pathway neurons, respectively. This study demonstrates the power of FLiP to detect changes in biochemical state induced by naturalistic experiences in behaving animals.