New and improved GRAB fluorescent sensors for monitoring dopaminergic activity in vivo

Abstract

The monoamine neuromodulator dopamine (DA) plays a critical role in the brain, and the 21 ability to directly measure dopaminergic activity is essential for understanding its 22 physiological functions. We therefore developed the first red fluorescent GPCR-activation-23 based DA (GRABDA) sensors and optimized versions of green fluorescent GRABDA sensors 24 following our previous studies. In response to extracellular DA, both the red and green 25 GRABDA sensors have a large increase in fluorescence (ΔF/F0 values of 150% and 340%, 26 respectively), with subcellular resolution, subsecond kinetics, and nanomolar to 27 submicromolar affinity. Moreover, both the red and green GRABDA sensors readily resolve 28 evoked DA release in mouse brain slices, detect compartmental DA release in live flies with 29 single-cell resolution, and report optogenetically elicited nigrostriatal DA release as well as 30 mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. 31 Importantly, co-expressing red GRABDA with either green GRABDA or the calcium indicator 32GCaMP6s provides a robust tool for simultaneously tracking neuronal activity and 33 dopaminergic signaling in distinct circuits in vivo. 35Dopamine (DA) is an essential monoamine neuromodulator produced primarily in the midbrain and 36 released throughout the central nervous system. A multitude of brain functions are regulated by DA, 37including motor control, motivation, learning and memory, and emotional control 1-9 . Consistent with 38these key physiological roles, altered DA signaling has been implicated in a variety of brain 39 disorders, including Parkinson's disease, addiction, schizophrenia, attention-deficit/hyperactivity 40 disorder, and posttraumatic stress disorder 10-20 . Thus, tools that can sense changes in DA 41 concentration with high spatiotemporal resolution, high specificity, and high sensitivity will greatly 42 facilitate our study of the diverse functions that the dopaminergic system plays under both 43 physiological and pathological conditions. 44Previous techniques for measuring DA dynamics, including microdialysis, electrochemical 45 probes, reporter cells, and gene expression-based assays, lack sufficient spatiotemporal resolution 46 and/or molecular specificity [21][22][23][24][25][26][27][28][29][30] . Recently, our group 31 and Patriarchi et al. 32 independently 47 developed two series of genetically encoded, G-protein-coupled receptor (GPCR)-based DA 48 sensors called GRABDA and dLight, respectively. Taking advantage of naturally occurring DA 49 receptors, these sensors convert a ligand-stabilized conformational change in the DA receptor into 50 an optical response via a conformation-sensitive fluorescent protein inserted in the receptor's third 51 intracellular loop. Our first-generation DA receptor-based sensors called GRABDA1m and 52 GRABDA1h were used to detect cell type-specific DA dynamics in several organisms, including 53 Drosophila, zebrafish, mice, and zebra finches 31,33-35 . Here, we employed semi-rational en...

A gradual backward shift of dopamine responses during associative learning

“…In the present study, we simply increased the delay duration between the cue and the reward (Figure 1). To detect dopamine dynamics, we injected adeno associated virus (AAV) in the VS to express the dopamine sensor GRAB DA2m (DA2m) 22 and measured fluorescence changes with fiber-fluorometry (Figure 1a, Supplemental Figure 1). Over multiple sessions of classical conditioning, naive mice gradually acquired anticipatory licking (Figure 1b).…”

“…In experiments of reversal learning and repeated learning, AAV5-CAG-FLEX-tdTomato (7.8 × 10 12 particles/ml; UNC Vector Core) and AAV5-CAG-FLEX-GCaMP7f were co-injected (1:1) in 3 DAT-cre mice and, AAV5-CAG-tdTomato (4.3 × 10 12 particles/ml; UNC Vector Core) and AAV5-TRE3G-GCaMP6f were co-injected (1:1) in 2 DAT-tTA mice. For expression of dopamine sensor in the VS, AAV9-hSyn-DA2m (1.01 × 10 13 ; ViGene bioscience) 22 was injected unilaterally in the VS (300nl, Bregma +1.45 AP, 1.4 ML, 4.35 DV) in 7 VGluT3-Cre/LDL-tdTomato mice. These mice with dopamine sensor were used for experiments of initial learning, and 3 of them were also used for reversal learning.…”

Neural and Hormonal Control of Sexual Behavior

“…Numerous groups have reported elevated dopamine release in the NAc upon presentation of a potential mate and during active mating in both males and females ( 164-172 ). With recent methodological advances enabling greater temporal resolution, we see escalating amount of dopamine release in a male as the mating behavioral suite progresses, with the highest release associated with ejaculation ( 173 , 174 ). In female rats, dopamine release is dependent on the testing environment.…”

Slowly evolving dopaminergic activity modulates the moment-to-moment probability of movement initiation

“…Similar ramping dynamics 137 and baseline differences were found in a dorsal lateral striatal "lick area" (DLS, Sippy et al, 2015), 138 in both the fluorescence of GCaMP6f in DAN axon terminals ( Figure 2B), as well as GRAB DA2m 139 (DA 2m ) expressed in striatal cells ( Figures 1F and 2C). DA 2m is a new, improved extracellular 140 dopamine indicator derived from the dopamine-2-receptor (Sun et al, 2020). Thus, ramping SNc 141…”