A genetically-encoded fluorescent sensor enables rapid and specific detection of dopamine in flies, fish, and mice

AbstractDopamine (DA) is a central monoamine neurotransmitter involved in many physiological and pathological processes. A longstanding yet largely unmet goal is to measure DA changes reliably and specifically with high spatiotemporal precision, particularly in animals executing complex behaviors. Here we report the development of novel genetically-encoded GPCR-Activation-Based-DA (GRABDA) sensors that enable these measurements. In response to extracellular DA rises, GRABDA sensors exhibit large fluorescence increases (ΔF/F0∼90%) with sub-second kinetics, nanomolar to sub-micromolar affinities, and excellent molecular specificity. Importantly, GRABDA sensors can resolve a single-electrical-stimulus evoked DA release in mouse brain slices, and detect endogenous DA release in the intact brains of flies, fish, and mice. In freely-behaving mice, GRABDA sensors readily report optogenetically-elicited nigrostriatal DA release and depict dynamic mesoaccumbens DA changes during Pavlovian conditioning or during sexual behaviors. Thus, GRABDA sensors enable spatiotemporal precise measurements of DA dynamics in a variety of model organisms while exhibiting complex behaviors.

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

10.1101/2020.03.28.013722 ◽

2020 ◽

Cited By ~ 5

Author(s):

Fangmiao Sun ◽

Jingheng Zhou ◽

Bing Dai ◽

Tongrui Qian ◽

Jianzhi Zeng ◽

...

Keyword(s):

Critical Role ◽

Brain Slices ◽

Dopaminergic Activity ◽

Gpcr Activation ◽

Calcium Indicator ◽

Freely Behaving ◽

Green Fluorescent ◽

Subcellular Resolution ◽

Da Release

The monoamine neuromodulator dopamine (DA) plays a critical role in the brain, and the ability to directly measure dopaminergic activity is essential for understanding its physiological functions. We therefore developed the first red fluorescent GPCR-activation–based DA (GRABDA) sensors and optimized versions of green fluorescent GRABDA sensors following our previous studies. In response to extracellular DA, both the red and green GRABDA sensors have a large increase in fluorescence (ΔF/F0 values of 150% and 340%, respectively), with subcellular resolution, subsecond kinetics, and nanomolar to submicromolar affinity. Moreover, both the red and green GRABDA sensors readily resolve evoked DA release in mouse brain slices, detect compartmental DA release in live flies with single-cell resolution, and report optogenetically elicited nigrostriatal DA release as well as mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. Importantly, co-expressing red GRABDA with either green GRABDA or the calcium indicator GCaMP6s provides a robust tool for simultaneously tracking neuronal activity and dopaminergic signaling in distinct circuits in vivo.

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GPCR-Based Dopamine Sensors—A Detailed Guide to Inform Sensor Choice for In Vivo Imaging

International Journal of Molecular Sciences ◽

10.3390/ijms21218048 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8048

Author(s):

Marie A. Labouesse ◽

Reto B. Cola ◽

Tommaso Patriarchi

Keyword(s):

In Vivo Imaging ◽

Dynamic Range ◽

New Techniques ◽

Experimental Parameters ◽

Sensor Properties ◽

Molecular Specificity ◽

Freely Behaving ◽

The Brain ◽

Da Release

Understanding how dopamine (DA) encodes behavior depends on technologies that can reliably monitor DA release in freely-behaving animals. Recently, red and green genetically encoded sensors for DA (dLight, GRAB-DA) were developed and now provide the ability to track release dynamics at a subsecond resolution, with submicromolar affinity and high molecular specificity. Combined with rapid developments in in vivo imaging, these sensors have the potential to transform the field of DA sensing and DA-based drug discovery. When implementing these tools in the laboratory, it is important to consider there is not a ‘one-size-fits-all’ sensor. Sensor properties, most importantly their affinity and dynamic range, must be carefully chosen to match local DA levels. Molecular specificity, sensor kinetics, spectral properties, brightness, sensor scaffold and pharmacology can further influence sensor choice depending on the experimental question. In this review, we use DA as an example; we briefly summarize old and new techniques to monitor DA release, including DA biosensors. We then outline a map of DA heterogeneity across the brain and provide a guide for optimal sensor choice and implementation based on local DA levels and other experimental parameters. Altogether this review should act as a tool to guide DA sensor choice for end-users.

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Function and regulation of TRPP2 at the plasma membrane

AJP Renal Physiology ◽

10.1152/ajprenal.90277.2008 ◽

2009 ◽

Vol 297 (1) ◽

pp. F1-F9 ◽

Cited By ~ 48

Author(s):

Leonidas Tsiokas

Keyword(s):

Cell Surface ◽

Transient Receptor Potential ◽

Fluid Shear Stress ◽

Ionic Currents ◽

Molecular Assembly ◽

Model Organisms ◽

Genetic Studies ◽

Naturally Occurring ◽

Gpcr Activation ◽

Extracellular Stimuli

The vast majority (∼99%) of all known cases of autosomal dominant polycystic kidney disease (ADPKD) are caused by naturally occurring mutations in two separate, but genetically interacting, loci, pkd1 and pkd2. pkd1 encodes a large multispanning membrane protein (PKD1) of unknown function, while pkd2 encodes a protein (TRPP2, polycystin-2, or PKD2) of the transient receptor potential (TRP) superfamily of ion channels. Biochemical, functional, and genetic studies support a model in which PKD1 physically interacts with TRPP2 to form an ion channel complex that conveys extracellular stimuli to ionic currents. However, the molecular identity of these extracellular stimuli remains elusive. Functional studies in cell culture show that TRPP2 can be activated in response to mechanical cues (fluid shear stress) and/or receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) activation at the cell surface. Recent genetic studies in Chlamydomonas reinhardtii show that CrPKD2 functions in a pathway linking cell-cell adhesion and Ca2+ signaling. The mode of activation depends on protein-protein interactions with other channel subunits and auxiliary proteins. Therefore, understanding the mechanisms underlying the molecular makeup of TRPP2-containing complexes is critical in delineating the mechanisms of TRPP2 activation and, most importantly, the mechanisms by which naturally occurring mutations in pkd1 or pkd2 lead not only to ADPKD, but also to other defects reported in model organisms lacking functional TRPP2. This review focuses on the molecular assembly, function, and regulation of TRPP2 as a cell surface cation channel and discusses its potential role in Ca2+ signaling and ADPKD pathophysiology.

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Sex-dependent changes in murine striatal dopamine release, sleep, and behavior during spontaneous Δ-9-tetrahydrocannabinol abstinence

10.1101/2021.12.01.470765 ◽

2021 ◽

Author(s):

Andrew J. Kesner ◽

Yolanda Mateo ◽

Karina P. Abrahao ◽

Stephanie Ramos-Maciel ◽

Matthew J. Pava ◽

...

Keyword(s):

Sleep Disturbances ◽

Animal Studies ◽

Brain Slices ◽

Withdrawal Symptoms ◽

Exposure Model ◽

Sleep Disruption ◽

Clinical Observations ◽

Induced Changes ◽

And Behavior ◽

Da Release

AbstractWithdrawal symptoms are observed upon cessation of cannabis use in humans. Although animal studies have examined withdrawal symptoms following exposure to delta-9-tetrahydrocannabinol (THC), difficulties in obtaining objective measures of spontaneous withdrawal using paradigms that mimic cessation of use in humans have slowed research. The neuromodulator dopamine (DA) is known to be affected by chronic THC treatment and plays a role in many behaviors related to human THC withdrawal symptoms. These symptoms include sleep disturbances that often drive relapse, and emotional behaviors, e.g., irritability and anhedonia. We examined THC withdrawal-induced changes in striatal DA release and the extent to which sleep disruption and behavioral maladaptation manifest during withdrawal in a mouse chronic cannabis exposure model. Using a THC treatment regimen known to produce tolerance we measured electrically elicited DA release in acute brain slices from different striatal subregions during early and late THC abstinence. Long-term polysomnographic recordings from mice were used to assess vigilance state and sleep architecture before, during, and after THC treatment. We additionally assessed how behaviors that model human withdrawal symptoms are altered by chronic THC treatment in early and late abstinence. We detected altered striatal DA release, sleep disturbances that mimic clinical observations, and behavioral maladaptation in mice following tolerance inducing THC treatment. Sex differences were observed in nearly all metrics. Altered striatal DA release, sleep and affect-related behaviors associated with spontaneous THC abstinence were more consistently observed in male mice. To our knowledge these findings provide the first model of directly translatable non-precipitated cannabis withdrawal symptoms, in particular, sleep disruption.

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Properties of Dopamine Release and Uptake in the Songbird Basal Ganglia

Journal of Neurophysiology ◽

10.1152/jn.01053.2004 ◽

2005 ◽

Vol 93 (4) ◽

pp. 1871-1879 ◽

Cited By ~ 28

Author(s):

Samuel D. Gale ◽

David J. Perkel

Keyword(s):

Basal Ganglia ◽

Brain Slices ◽

Vocal Learning ◽

Monoamine Transporters ◽

Anterior Forebrain ◽

Song Production ◽

Electrode Selectivity ◽

Area X ◽

Da Release

Vocal learning in songbirds requires a basal ganglia circuit termed the anterior forebrain pathway (AFP). The AFP is not required for song production, and its role in song learning is not well understood. Like the mammalian striatum, the striatal component of the AFP, Area X, receives dense dopaminergic innervation from the midbrain. Since dopamine (DA) clearly plays a crucial role in basal ganglia–mediated motor control and learning in mammals, it seems likely that DA signaling contributes importantly to the functions of Area X as well. In this study, we used voltammetric methods to detect subsecond changes in extracellular DA concentration to gain better understanding of the properties and regulation of DA release and uptake in Area X. We electrically stimulated Ca2+- and action potential–dependent release of an electroactive substance in Area X brain slices and identified the substance as DA by the voltammetric waveform, electrode selectivity, and neurochemical and pharmacological evidence. As in the mammalian striatum, DA release in Area X is depressed by autoinhibition, and the lifetime of extracellular DA is strongly constrained by monoamine transporters. These results add to the known physiological similarities of the mammalian and songbird striatum and support further use of voltammetry in songbirds to investigate the role of basal ganglia DA in motor learning.

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Differences in Nicotine Encoding Dopamine Release between the Striatum and Shell Portion of the Nucleus Accumbens

Cell Transplantation ◽

10.1177/0963689718775382 ◽

2018 ◽

Vol 28 (3) ◽

pp. 248-261 ◽

Cited By ~ 4

Author(s):

Yuan-Hao Chen ◽

Bon-Jour Lin ◽

Tsung-Hsun Hsieh ◽

Tung-Tai Kuo ◽

Jonathan Miller ◽

...

Keyword(s):

Nucleus Accumbens ◽

High Frequency ◽

Dopamine Release ◽

Brain Slices ◽

Dorsal Striatum ◽

High Frequency Stimulation ◽

Sprague Dawley ◽

Frequency Stimulation ◽

Da Release

The aim of this work was to determine the effect of nicotine desensitization on dopamine (DA) release in the dorsal striatum and shell of the nucleus accumbens (NAc) from brain slices. In vitro fast-scan cyclic voltammetry analysis was used to evaluate dopamine release in the dorsal striatum and the NAc shell of Sprague–Dawley rats after infusion of nicotine, a nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine (Mec), and an α4β2 cholinergic receptor antagonist (DHβe). DA release related to nicotine desensitization in the striatum and NAc shell was compared. In both structures, tonic release was suppressed by inhibition of the nicotine receptor (via Mec) and the α4β2 receptor (via DHβe). Paired-pulse ratio (PPR) was facilitated in both structures after nicotine and Mec infusion, and this facilitation was suppressed by increasing the stimulation interval. After variable frequency stimulation (simulating phasic burst), nicotine infusion induced significant augmentation of DA release in the striatum that was not seen in the absence of nicotine. In contrast, nicotine reduced phasic DA release in NAc, although frequency augmentation was seen both with and without nicotine. Evaluation of DA release evoked by various trains (high-frequency stimulation (HFS) 100 Hz) of high-frequency stimulation revealed significant enhancement after a train of three or more pulses in the striatum and NAc. The concentration differences between tonic and phasic release related to nicotine desensitization were more pronounced in the NAc shell. Nicotine desensitization is associated with suppression of tonic release of DA in both the striatum and NAc shell that may occur via the α4β2 subtype of nAChR, whereas phasic frequency-dependent augmentation and HFS-related gating release is more pronounced in the striatum than in the NAc shell. Differences between phasic and tonic release associated with nicotine desensitization may underlie processing of reward signals in the NAc shell, and this may have major implications for addictive behavior.

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Implication of synaptotagmins 4 and 7 in activity-dependent somatodendritic dopamine release

10.1101/2021.01.25.427983 ◽

2021 ◽

Author(s):

Benoît Delignat-Lavaud ◽

Charles Ducrot ◽

Willemieke Kouwenhoven ◽

Nina Feller ◽

Louis-Éric Trudeau

Keyword(s):

Molecular Mechanism ◽

Dopamine Release ◽

Brain Slices ◽

Calcium Sensitivity ◽

Knock Out ◽

Axon Terminals ◽

Calcium Sensors ◽

6 Hydroxydopamine ◽

Activity Dependent ◽

Da Release

ABSTRACTDopamine (DA) neurons can release DA not just from axon terminals, but also from their somatodendritic (STD) compartment thought a mechanism that is still incompletely understood. Using voltammetry in mouse mesencephalic brain slices, we find that STD DA release has low capacity, is stable in response to electrical but not optogenetic train pulses and shows a calcium sensitivity that is comparable to that of axonal release. It is also strikingly more resilient compared to axonal release in a 6‐ hydroxydopamine model of Parkinson’s disease plasticity. We find that the molecular mechanism of STD DA release differs from axonal release with regards to the implication of synaptotagmin (Syt) calcium sensors. While individual constitutive knock-out Syt4 and Syt7 is not sufficient to reduce STD DA release, removal of both isoforms reduces this release by ~50%, leaving axonal release unimpaired. Our works unveils clear differences in the mechanisms of STD and axonal DA release.

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L-type calcium channel contribution to striatal dopamine release is governed by calbindin-D28K, the dopamine transporter, D2-receptors, α2δ-subunits and sex differences

10.1101/2020.07.03.186411 ◽

2020 ◽

Author(s):

Katherine R. Brimblecombe ◽

Natalie Connor-Robson ◽

Bradley M. Roberts ◽

Caitlin Gracie ◽

Rebecca te Water Naude ◽

...

Keyword(s):

Brain Slices ◽

Metabolic Stress ◽

D2 Receptors ◽

Pacemaker Activity ◽

Fast Scan Cyclic Voltammetry ◽

Nigrostriatal Dopamine ◽

Neuroprotective Strategies ◽

Positive Regulators ◽

Calbindin D28k ◽

Da Release

AbstractCa2+ entry to nigrostriatal dopamine (DA) neurons and axons via L-type voltage-gated Ca2+ channels (LTCCs) contributes to pacemaker activity and DA release, but burdens cells with a metabolic stress promoting their vulnerability to parkinsonian degeneration. The level of LTCC function varies between subtypes of DA neurons, but is not proportional to LTCC expression level, indicating that LTCC function is governed by other factors. We used fast-scan cyclic voltammetry in mouse brain slices to identify mechanisms that govern whether LTCCs contribute to DA release in dorsal and ventral striatum. We find that calbindin-D28K limits LTCC function in a regionally and sexually divergent manner; D2-receptors and DA transporters are negative and positive regulators respectively; and lastly, that targeting α2δ subunits with gabapentinoid drugs restricts LTCC function without compromising DA release. These data reveal that LTCC function in DA axons is dynamically and locally regulated, which may prove useful for future neuroprotective strategies.

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A fluorescent sensor for spatiotemporally resolved endocannabinoid dynamics in vitro and in vivo

10.1101/2020.10.08.329169 ◽

2020 ◽

Author(s):

Ao Dong ◽

Kaikai He ◽

Barna Dudok ◽

Jordan S Farrell ◽

Wuqiang Guan ◽

...

Keyword(s):

Membrane Trafficking ◽

Basolateral Amygdala ◽

Fluorescent Sensor ◽

Brain Slices ◽

High Specificity ◽

Cultured Neurons ◽

Spatiotemporal Resolution ◽

Wide Range

Endocannabinoids (eCBs) are retrograde neuromodulators that play an important role in a wide range of physiological processes; however, the release and in vivo dynamics of eCBs remain largely unknown, due in part to a lack of suitable probes capable of detecting eCBs with sufficient spatiotemporal resolution. Here, we developed a new eCB sensor called GRABeCB2.0. This genetically encoded sensor consists of the human CB1 cannabinoid receptor fused to circular-permutated EGFP, providing cell membrane trafficking, second-resolution kinetics, high specificity for eCBs, and a robust fluorescence response at physiological eCB concentrations. Using the GRABeCB2.0 sensor, we monitored evoked changes in eCB dynamics in both cultured neurons and acute brain slices. Interestingly, in cultured neurons we also observed spontaneous compartmental eCB transients that spanned a distance of approximately 11 μm, suggesting constrained, localized eCB signaling. Moreover, by expressing GRABeCB2.0 in the mouse brain, we readily observed foot shock-elicited and running-triggered eCB transients in the basolateral amygdala and hippocampus, respectively. Lastly, we used GRABeCB2.0 in a mouse seizure model and observed a spreading wave of eCB release that followed a Ca2+ wave through the hippocampus. Thus, GRABeCB2.0 is a robust new probe for measuring the dynamics of eCB release under both physiological and pathological conditions.

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Spontaneous Formation of Melanin from Dopamine in the Presence of Iron

Antioxidants ◽

10.3390/antiox9121285 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1285

Author(s):

David M. Hedges ◽

Jordan T. Yorgason ◽

Andrew W. Perez ◽

Nathan D. Schilaty ◽

Benjamin M. Williams ◽

...

Keyword(s):

Oxidative Stress ◽

Amino Acids ◽

Hydrogen Peroxide ◽

Cyclic Voltammetry ◽

Brain Slices ◽

Fast Scan Cyclic Voltammetry ◽

Spontaneous Formation ◽

Ir Spectrophotometry ◽

Da Release

Parkinson’s disease is associated with degeneration of neuromelanin (NM)-containing substantia nigra dopamine (DA) neurons and subsequent decreases in striatal DA transmission. Dopamine spontaneously forms a melanin through a process called melanogenesis. The present study examines conditions that promote/prevent DA melanogenesis. The kinetics, intermediates, and products of DA conversion to melanin in vitro, and DA melanogenesis under varying levels of Fe3+, pro-oxidants, and antioxidants were examined. The rate of melanogenesis for DA was substantially greater than related catecholamines norepinephrine and epinephrine and their precursor amino acids tyrosine and l-Dopa as measured by UV-IR spectrophotometry. Dopamine melanogenesis was concentration dependent on the pro-oxidant species and Fe3+. Melanogenesis was enhanced by the pro-oxidant hydrogen peroxide (EC50 = 500 μM) and decreased by the antioxidants ascorbate (IC50 = 10 μM) and glutathione (GSH; IC50 = 5 μM). Spectrophotometric results were corroborated by tuning a fast-scan cyclic voltammetry system to monitor DA melanogenesis. Evoked DA release in striatal brain slices resulted in NM formation that was prevented by GSH. These findings suggest that DA melanogenesis occurs spontaneously under physiologically-relevant conditions of oxidative stress and that NM may act as a marker of past exposure to oxidative stress.

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