Monitoring Behaviorally Induced Biochemical Changes Using Fluorescence Lifetime Photometry

Abstract

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.

Cell-type specific asynchronous modulation of PKA by dopamine during reward based learning

“…Phosphorylation of the sensor by PKA induces a conformational change that increases FRET efficiency between the donor and the acceptor, resulting in faster fluorescence decay of the donor. Therefore, as previously validated in vitro 31,33 and in vivo 32 , a reduction (negative change) in fluorescence lifetime of FLIM-AKAR represents an increase in the net phosphorylation of PKA substrates in cells expressing the sensor.…”

“…This could be due to 1) incomplete blockade of PKA activity in either classes of SPNs 2) PKA independent cellular plasticity and/or 3) other brain regions involved in learning that may compensate for the function of NAc. Possibility 1 is less likely given we have previously observed a near complete blockade in PKA activation by PKI in both in vitro 31 and in vivo 32 . In addition, D2R-SPN PKA was strongly activated during reward omission trials, but PKA inhibition in D2R-SPNs did not have a significant effect on the extinction sessions.…”

“…To monitor SPN PKA activity in vivo, we performed fluorescence lifetime photometry of FLIM-AKAR through an optical fiber implanted in the NAc 32 . Briefly, FLIM-AKAR is a Förster resonance energy transfer (FRET)-based PKA activity reporter consisting of a donor fluorophore and a dark acceptor 31 .…”

“…Given the separate modulation of D1R-and D2R-SPN PKA activity during learning, we investigated if a selective inhibition of PKA activity in each neuron class differentially affects learning. To achieve celltype specific inhibition of PKA activity, we virally overexpressed PKIα, an endogenous inhibitory peptide for PKA, which, we previously showed, effectively blocks PKA activation in vitro 31 and in vivo 32 . We injected AAV1-FLEX-PKIalpha-IRES-nls-mRuby2 or AAV1-Cag-FLEX-eGFP (control) into Drd1a-Cre and Adora2a-Cre mice to selectively inhibit PKA in D1R-and D2R-SPNs, respectively ( Fig.…”

“…Simultaneous measurements of VTA DA neuron bulk Ca 2+ levels (referred as DA neuron activity below for simplicity) and NAc DA levels by multi-channel fiber photometry indicate that they are correlated with each other and consistent with RPE. Using fluorescence lifetime photometry (FLiP), which permits dynamic measurement of intracellular biochemical signals in genetically-defined neurons using a PKA substrate sensor (FLIM-AKAR) 31 , we monitored endogenous net PKA activity (the balance between PKA and phosphatase activity) in D1R-and D2R-expressing SPNs (D1R-SPNs and D2R-SPNs, respectively) 32 . We reveal dynamic changes in D1R-SPN and D2R-SPN PKA activity that are asynchronously and differentially triggered during reinforcement learning.…”