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The Role of the Dopamine Transporter in the Effects of Amphetamine on Sleep and Sleep Architecture in Drosophila

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Abstract

The dopamine transporter (DAT) mediates the inactivation of released dopamine (DA) through its reuptake, and thereby plays an important homeostatic role in dopaminergic neurotransmission. Amphetamines exert their stimulant effects by targeting DAT and inducing the reverse transport of DA, leading to a dramatic increase of extracellular DA. Animal models have proven critical to investigating the molecular and cellular mechanisms underlying transporter function and its modulation by psychostimulants such as amphetamine. Here we establish a behavioral model for amphetamine action using adult Drosophila melanogaster. We use it to characterize the effects of amphetamine on sleep and sleep architecture. Our data show that amphetamine induces hyperactivity and disrupts sleep in a DA-dependent manner. Flies that do not express a functional DAT (dDAT null mutants) have been shown to be hyperactive and to exhibit significantly reduced sleep at baseline. Our data show that, in contrast to its action in control flies, amphetamine decreases the locomotor activity of dDAT null mutants and restores their sleep by modulating distinct aspects of sleep structure. To begin to explore the circuitry involved in the actions of amphetamine on sleep, we also describe the localization of dDAT throughout the fly brain, particularly in neuropils known to regulate sleep. Together, our data establish Drosophila as a robust model for studying the regulatory mechanisms that govern DAT function and psychostimulant action.

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Acknowledgements

This work was supported by R01 DA041510 (JAJ and CSK), U01 DA042233 (JAJ, CSK and BLW), and T32 MH018870 (SKJ). We thank Dr. Eric Gouaux for providing the anti-dDAT antibody, Dr. Stephen Thornquist for advice on using the antibody for fluorescence imaging, Dr. Claudia Ma for her help with the initial experimental setup, and Dr. Mimi Shirazu-Hiza for sharing her Trikinetic monitors during the initial setup of the assay.

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Author notes

  1. Caline S. Karam and Brenna L. Williams have contributed equally.

Authors and Affiliations

  1. Department of Psychiatry, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA

    Caline S. Karam, Sandra K. Jones & Jonathan A. Javitch

  2. Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA

    Caline S. Karam, Brenna L. Williams, Sandra K. Jones & Jonathan A. Javitch

  3. Department of Pharmacology, Columbia University Vagelos College of Physicians and Surgeons, 1051 Riverside Dr, Unit 19, New York, NY, 10032, USA

    Jonathan A. Javitch

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  1. Caline S. Karam
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  4. Jonathan A. Javitch
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Contributions

CSK and JAJ designed the experiments. CSK, BLW, SKJ, and JAJ analyzed the data, discussed the results, and wrote the manuscript. CSK developed the behavioral assay and conducted the behavioral experiments. BLW performed the analysis of activity, sleep, and sleep structure data, in addition to all statistical analyses. BLW and SKJ performed the immunostaining and imaging analysis.

Corresponding author

Correspondence to Jonathan A. Javitch.

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Supplementary Information

Below is the link to the electronic supplementary material.

11064_2021_3275_MOESM1_ESM.jpg

Supplementary file1 (JPG 1046 KB)

Supplementary Image 1. Maximum intensity projection of neuronal cell bodies, as well as parts of the PCB and IB, in whole adult fly brains stained with anti-dDAT antibody (green), imaged under 63 × with a 3.5 × optical zoom. Scale bar indicates 10 µm. LIGHTNING adaptive deconvolution was used to obtain maximum resolution [90].

Supplementary file2 (MP4 67941 KB)

Supplementary Video 1. 3D reconstruction of an anterior segment consisting of 122 sections of 0.3-µm thickness from a whole-mount Drosophila brain fluorescently labeled with anti-dDAT antibody (green), captured by confocal microscopy at 63 ×.

Supplementary file3 (MP4 105947 KB)

Supplementary Video 2. 3D reconstruction of a posterior segment consisting of 175 sections of 0.3-µm thickness from a whole-mount Drosophila brain fluorescently labeled with anti-dDAT antibody (green), captured by confocal microscopy at 63 ×.

Supplementary file4 (MP4 20588 KB)

Supplementary Video 3. Raw fluorescent Z-stack images showing antibody-labeled dDAT (green) in Drosophila adult brain captured at 40 ×.

Supplementary file5 (MP4 112137 KB)

Supplementary Video 4. Raw fluorescent Z-stack images showing antibody-labeled dDAT (green) captured at 63 ×.

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Karam, C.S., Williams, B.L., Jones, S.K. et al. The Role of the Dopamine Transporter in the Effects of Amphetamine on Sleep and Sleep Architecture in Drosophila. Neurochem Res 47, 177–189 (2022). https://doi.org/10.1007/s11064-021-03275-4

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  • DOI: https://doi.org/10.1007/s11064-021-03275-4

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