Silencing of Syntaxin 1A in the Dopaminergic Neurons Decreases the Activity of the Dopamine Transporter and Prevents Amphetamine-Induced Behaviors in C. elegans

Lanzo, Ambra; Safratowich, Bryan D.; Kudumala, Sirisha; Gallotta, Ivan; Zampi, Giordano; Schiavi, Elia Di; Carvelli, Lucia

doi:10.3389/fphys.2018.00576

Cited by 9 publications

(14 citation statements)

References 49 publications

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“…1a). These results confirm our previously published data showing that amphetamine causes SWIP in C. elegans [Carvelli et al, 2010;Safratowich et al, 2013;Lanzo et al, 2018]. Following the first exposure to the amphetamine or control solution, the animals were reallocated to food-seeded plates after being briefly immerged (∼1 min) in wells containing water to ensure that no residue of amphetamine was left on the animals' bodies.…”

Section: Repeated Exposure To Amphetamine Reduces Amphetamine-induced Behaviorssupporting

confidence: 88%

Caenorhabditis elegans as an in vivo Model to Assess Amphetamine Tolerance

et al. 2020

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Amphetamine is a potent psychostimulant also used to treat attention deficit/hyperactivity disorder and narcolepsy. In vivo and in vitro data have demonstrated that amphetamine increases the amount of extra synaptic dopamine by both inhibiting reuptake and promoting efflux of dopamine through the dopamine transporter. Previous studies have shown that chronic use of amphetamine causes tolerance to the drug. Thus, since the molecular mechanisms underlying tolerance to amphetamine are still unknown, an animal model to identify the neurochemical mechanisms associated with drug tolerance is greatly needed. Here we took advantage of a unique behavior caused by amphetamine in Caenorhabditis elegans to investigate whether this simple, but powerful, genetic model develops tolerance following repeated exposure to amphetamine. We found that at least 3 treatments with 0.5 mM amphetamine were necessary to see a reduction in the amphetamine-induced behavior and, thus, to promote tolerance. Moreover, we found that, after intervals of 60/90 minutes between treatments, animals were more likely to exhibit tolerance than animals that underwent 10-minute intervals between treatments. Taken together, our results show that C. elegans is a suitable system to study tolerance to drugs of abuse such as amphetamines.

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Section: Repeated Exposure To Amphetamine Reduces Amphetamine-induced Behaviorssupporting

confidence: 88%

Caenorhabditis elegans as an in vivo Model to Assess Amphetamine Tolerance

et al. 2020

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“…Cell amperometry and patch-clamp electrophysiology: Cells were washed three times with Lub's external (130 mM NaCl, 10 mM HEPES, 34 mM dextrose, 1.5 mM CaCl 2 , 0.5 mM MgSO 4 , 1.3 mM KH 2 PO 4 , pH 7. 35 and 300-310 mOsms/L) bath solution at room temperature. The internal solution for the whole-cell recording contained 120 mM KCl, 10mM NaCl 0.1 mM CaCl 2 , 2 mM MgCl 2 , 1.1 mM EGTA, 10 mM Hepes, and 30 mM dextrose plus DA (2 mM or as specified in the text) adjusted to pH 7.…”

Section: Dna Constructsmentioning

confidence: 99%

“…The internal solution for the whole-cell recording contained 120 mM KCl, 10mM NaCl 0.1 mM CaCl 2 , 2 mM MgCl 2 , 1.1 mM EGTA, 10 mM Hepes, and 30 mM dextrose plus DA (2 mM or as specified in the text) adjusted to pH 7. 35. Patch electrodes were pulled from quartz pipettes on a P-2000 puller (Sutter Instruments, Novato, CA) and filled with the internal solution.…”

Section: Dna Constructsmentioning

confidence: 99%

“…Syntaxin 1A (Stx1) is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein that plays a key role in vesicular synaptic release 29 and is regulated by Munc18-1 binding [30][31] . In addition to its role in vesicular fusion, Stx1 interacts with and regulates the function of transmembrane proteins, including ion channels and transporters 12,[32][33][34][35][36][37] . Stx1 directly interacts with the DAT N-terminus within the first 33 amino acids 33 .…”

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confidence: 99%

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Syntaxin1 Ser14 Phosphorylation is Required for Non-Vesicular Dopamine Release

Shekar

Mabry

Cheng

et al. 2022

Preprint

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Amphetamine (AMPH), a psychostimulant commonly prescribed for the treatment of neuropsychiatric and neurological disorders, has a high liability for abuse. The abuse and psychomotor stimulant properties of AMPH are primarily associated with its ability to increase dopamine (DA) neurotransmission. This increase is mediated, in large part, by non-vesicular DA release (DA efflux). DA efflux is the result of reversal of the DA transporter (DAT) promoted by AMPH. Syntaxin 1 (Stx1) is a SNARE protein that plays a pivotal role in vesicular release. Previously, we have shown that Stx1 also interacts with the distal DAT N-terminus, an event promoted by AMPH. Stx1 is phosphorylated at Ser14 by casein kinase II (CK2). Using Drosophila Melanogaster as an animal model, we show that this phosphorylation event is critical for non-vesicular DA release and regulates the expression of AMPH preference as well as the ability of AMPH to promote mating drive. We also show that reverse transport of DA mediated by DAT underlies these complex behaviors promoted by AMPH. Our molecular dynamics (MD) simulations of the phosphorylated DAT/Stx1 complex demonstrate that the phosphorylation state of these proteins plays a key role in allowing DAT to dwell in an efflux-willing state. This state also supports constitutive DA efflux (CDE), an event that occurs in the absence of AMPH. The DAT-Stx1 phosphorylated complex is characterized by the breakdown of two key salt bridges in DAT, K66-D345 and E428-R445, which are critical for the formation of the intracellular (IC) gate and for transport function. The breaking of these salt bridges leads to an opening and hydration of the DAT intracellular vestibule, allowing DA to bind from the cytosol, a mechanism that we hypothesize leads to CDE. We further determine the importance of Stx1 phosphorylation in CDE by pharmacologically inhibiting CK2 with CX-4945, a molecule currently in phase II clinical trials for cancer treatment. CX-4945 treatment prevented the expression of CDE in isolated Drosophila Melanogaster brains as well as behaviors associated with CDE. Thus, our results suggest that Stx1 phosphorylation is a possible pharmacological target for the treatment of AMPH abuse.

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“…On the other hand, pharmacological or genetic manipulations averting synthesis and release of DA and blocking DOP-3 receptor function prevent SWIP 6 . Taken together, these already published data have established SWIP as a reliable tool I) to study the behavioral effects caused by mutated proteins within dopaminergic synapses 3,4,7 and II) to be employed for forward genetic screens for the identification of novel regulatory pathways involved in DA signaling [7][8][9][10][11][12] . Additionally, by providing an easily quantifiable readout of drug-induced behavior in living animals, SWIP enables the elucidation of mechanisms of action of drugs like amphetamine (AMPH) and azaperone at the dopaminergic synapses 5,6,[13][14][15] .…”

Section: Introductionmentioning

confidence: 96%

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

Kudumala

Sossi

Carvelli

2019

JoVE

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The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similarly to mammals, dopamine (DA) controls several functions in C. elegans including learning and motor activity. Conditions that stimulate DA release, e.g. amphetamine (AMPH) treatments, or that prevent DA clearance, e.g. animals lacking the DA transporter (dat-1) which are incapable of reaccumulating DA into the neurons, generate an excess of extracellular DA ultimately resulting in inhibited locomotion. This behavior is particularly evident when animals swim in water. In fact, while wild-type animals continue to swim for an extended period, dat-1 null mutants and wild-type treated with AMPH or inhibitors of the DA transporter sink to the bottom of the well and do not move. This behavior is termed "Swimming Induced Paralysis" (SWIP). Although SWIP assay is well established, a detailed description of the method is lacking. Here we describe a step-by-step guide to perform SWIP. To perform the assay, late larval stage-4 animals are placed in a glass spot plate containing control sucrose solution with or without AMPH. Animals are scored for their swimming behavior either manually by visualization under a stereoscope or automatically by recording with a camera mounted on the stereoscope. Videos are then analyzed using a tracking software, which yields a visual representation of thrashing frequency and paralysis in the form of heat maps. Both the manual and automated systems guarantee an easily quantifiable readout of the animals' swimming ability and thus facilitate screening for animals bearing mutations within the dopaminergic system or for auxiliary genes. In addition, SWIP can be used to elucidate the mechanism of action of drugs of abuse such as AMPH.

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Silencing of Syntaxin 1A in the Dopaminergic Neurons Decreases the Activity of the Dopamine Transporter and Prevents Amphetamine-Induced Behaviors in C. elegans

Cited by 9 publications

References 49 publications

<b><i>Caenorhabditis elegans</i></b> as an in vivo Model to Assess Amphetamine Tolerance

<b><i>Caenorhabditis elegans</i></b> as an in vivo Model to Assess Amphetamine Tolerance

Syntaxin1 Ser14 Phosphorylation is Required for Non-Vesicular Dopamine Release

Swimming Induced Paralysis to Assess Dopamine Signaling in <em>Caenorhabditis elegans</em>

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