Neuronal Imaging of Dopamine Dynamics

 

Science  29 Jun 2018: Vol. 360, Issue 6396, eaat4422

Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors

Tommaso Patriarchi, et.al.

Department of Biochemistry and Molecular Medicine, University of California, Davis, 2700 Stockton Boulevard, Sacramento, CA 95817, USA.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

Waitt Advanced Biophotonics Center, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

Department of Neurobiology, Northwestern University, Evanston, IL 60208, USA.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94131, USA.

Vollum Institute, Oregon Health & Science University, Portland, OR 97239, USA.

 [paraphrase]

Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires spatiotemporally precise measurement of neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in mouse striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in mouse cortex. We also validated our sensor design platform for developing norepinephrine,    serotonin,    melatonin,    and opioid neuropeptide indicators.

Animal behavior is influenced by the release of neuromodulators such as dopamine (DA), which signal behavioral variables that are relevant to the functioning of circuits brainwide. Projections from dopaminergic nuclei to the striatum and cortex, for example, play important roles in reinforcement learning, decision-making, and motor control. Loss of DA or dysfunction of its target circuits has been linked to disorders such as Parkinson’s disease,    schizophrenia,     and addiction.

Much work has been devoted to determining how neural representations of behavioral states are encoded in the firing patterns of neuromodulatory neurons, but very little is known about how the precise release of neuromodulators alters the function of their target circuits. To address this problem, an essential step is to monitor the spatiotemporal dynamics of neuromodulatory signals in target circuits while also measuring and manipulating the elements of the circuit during behavior.

Analytical techniques such as microdialysis and electrochemical microsensors have provided useful insights about neuromodulator presence but suffer from poor spatial and/or temporal resolution and cannot be targeted to cells of interest. Optical approaches such as injected cell-based systems (CNiFERs) and reporter gene–based iTango can reveal DA release with high molecular specificity. However, these systems are limited by poor temporal resolution (seconds to hours), preventing direct detection of DA release events that occur on a subsecond time scale.

High-quality single fluorescence protein (FP)–based sensors that report calcium or glutamate transients with subsecond temporal resolution have recently been developed and are widely used. Here, we report the development of a set of single FP–based DA sensors, named dLight1, that enables imaging of DA transients with high spatiotemporal resolution in behaving animals.

We developed and applied a new class of genetically encoded indicators that overcome major barriers of current methods to permit high-resolution imaging of DA dynamics in acute brain slices and in behaving mice. The submicromolar affinity and fast kinetics of dLight1 offer fast temporal resolution (10 ms on, 100 ms off) to detect the physiologically or behaviorally relevant DA transients with higher molecular specificity relative to existing electrochemical or cell-based probes. For example, in NAc of freely behaving mice, longitudinal measurements revealed different changes in time-resolved DA signals encoding either predictive cue or reward consumption across learning.

The disparate contributions of synaptic,    extrasynaptic,    and spillover DA events to circuit function are not addressable without fast, robust, and genetically encoded sensors. In a dorsal striatal slice, dLight1 reliably detected the concentration and time course of DA transients and their modifications by pharmacological compounds. The rapid rise of fluorescence (10 ms) and the peak concentration (10 to 30 μM) of DA after electrical stimulation indicates that the initial measures of DA are closely associated with the site of release. The decline of fluorescence, particularly in the presence of cocaine, results primarily from reuptake and diffusion of DA    away from release sites.

dLight1 also permits measurement of functionally heterogeneous DA transients at the cellular level with high spatial resolution. In the cortex, two-photon imaging with dLight1 revealed a DA transient map with spatially distributed, functionally heterogeneous DA signals during a visuomotor learning task. Simultaneous calcium imaging can further determine how spatiotemporal differences in DA levels relate to ongoing neural activity and influence associative learning or goal-directed behavior.

dLight1.1 and dLight1.2 are optimized sensor variants that can be immediately applied to ex vivo or in vivo studies, as they offer a good balance between dynamic range and affinity. Other dLight variants may be suitable for measuring synaptic release (dLight1.3) or tonic DA transients (dLight1.4). Given the broadly tunable affinity and dynamic range of dLight1, protein engineering and high-throughput screening efforts can further optimize the signal-to-noise ratio and molecular specificity as well as the performance of other neuromodulator indicators.

In combination with calcium imaging and optogenetics, our sensors are well poised to permit direct functional analysis of how the spatiotemporal coding of neuromodulatory signaling mediates the plasticity and function of target circuits.

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