Abstract
The dopamine transporter (DAT) plays a fundamental role in dopamine (DA) neurotransmission by retrieving the transmitter from the extracellular space back into the DA nerve terminal. Several lines of evidence suggest that in addition to uptake or forward transport, DAT can also function to release DA. This process, which is referred to as DAT-mediated release or efflux, is the mechanism used by potent and highly addictive psychostimulants, such as amphetamine (AMPH) and its analogues, to increase extracellular DA levels in motivational and reward areas of the brain. Conventionally, dopaminergic neurons release DA through a calcium-dependent exocytotic vesicular process. In the past several years, our lab and others have identified signaling pathways that promote DA release through DAT. In this review chapter and step-by-step protocol, we will describe assays to study endogenous DA efflux and DAT dynamics using acute brain slices combined with high-performance liquid chromatography (HPLC) for DA detection along with a modification of a classically used radioactive uptake assay to measure levels of DA uptake. Further examining DAT functions and DA dynamics is key for the development of novel therapeutics in DA-related disorders including substance abuse. Here, we described the use of acute brain slices further dissected into precise regions of interest to examine the dual function of DAT.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Zahm DS, Trimble M (2008) The dopaminergic projection system, basal forebrain macrosystems, and conditioned stimuli. CNS Spectr 13:32ā40
Giros B, Caron MG (1993) Molecular characterization of the dopamine transporter. Trends Pharmacol Sci 14:43ā49. https://doi.org/10.1016/0165-6147(93)90029-J
Volkow ND, Fowler JS, Wang GJ, Goldstein RZ (2002) Role of dopamine, the frontal cortex and memory circuits in drug addiction: insight from imaging studies. Neurobiol Learn Mem 78:610ā624
Di Chiara G (2002) Nucleus accumbens shell and core dopamine: differential role in behavior and addiction. Behav Brain Res 137:75ā114. https://doi.org/10.1016/S0166-4328(02)00286-3
Wanat MJ, Willuhn I, Clark JJ, Phillips PEM (2009) Phasic dopamine release in appetitive behaviors and drug addiction. Curr Drug Abuse Rev 2(2):195ā213. https://doi.org/10.2174/1874473710902020195
Volkow ND, Wang GJ, Newcorn J et al (2007) Brain dopamine transporter levels in treatment and drug naĆÆve adults with ADHD. NeuroImage 34:1182ā1190. https://doi.org/10.1016/j.neuroimage.2006.10.014
Leenders KL, Palmer AJ, Quinn N et al (1986) Brain dopamine metabolism in patients with Parkinsonās disease measured with positron emission tomography. J Neurol Neurosurg Psychiatry 49:853ā860. https://doi.org/10.1136/jnnp.49.8.853
Scherman D, Desnos C, Darchen F et al (1989) Striatal dopamine deficiency in Parkinsonās disease: role of aging. Ann Neurol 26:551ā557. https://doi.org/10.1002/ana.410260409
Aggarwal S, Mortensen O (2017) Overview of monoamine transporters. Curr Protoc Pharmacol 79:12.16.1ā12.16.17. https://doi.org/10.1002/CPPH.32
Nirenberg MJ, Vaughan RA, Uhl GR et al (1996) The dopamine transporter is localized to dendritic and axonal plasma membranes of nigrostriatal dopaminergic neurons. J Neurosci 16:436ā447. https://doi.org/10.1523/jneurosci.16-02-00436.1996
Blanchard V, Raisman-Vozari R, Vyas S et al (1994) Differential expression of tyrosine hydroxylase and membrane dopamine transporter genes in subpopulations of dopaminergic neurons of the rat mesencephalon. Mol Brain Res 22:29ā38. https://doi.org/10.1016/0169-328X(94)90029-9
Hersch SM, Yi H, Heilman CJ, Edwards RH, Levey AI (1997) Subcellular localization and molecular topology of the dopamine transporter in the striatum and substantia nigra. J Comp Neurol 388(2):211ā227
Patel JC, Witkovsky P, Avshalumov MV, Rice ME (2009) Mobilization of calcium from intracellular stores facilitates somatodendritic dopamine release. J Neurosci 29:6568ā6579. https://doi.org/10.1523/JNEUROSCI.0181-09.2009
Ritz MC, Lamb RJ, Goldberg SR, Kuhar MJ (1987) Cocaine receptors on dopamine transporters are related to self-administration of cocaine. Science (80- ) 237:1219ā1223. https://doi.org/10.1126/science.2820058
Fischer JF, Cho AK (1979) Chemical release of dopamine from striatal homogenates: evidence for an exchange diffusion model. J Pharmacol Exp Ther 208:203ā209
Kahlig KM, Binda F, Khoshbouei H et al (2005) Amphetamine induces dopamine efflux through a dopamine transporter channel. Proc Natl Acad Sci U S A 102:3495ā3500. https://doi.org/10.1073/pnas.0407737102
Mauna JC, Harris SS, Pino JA et al (2019) G protein Ī²Ī³ subunits play a critical role in the actions of amphetamine. Transl Psychiatry 9:1ā11. https://doi.org/10.1038/s41398-019-0387-8
Sulzer D, Maidment NT, Rayport S (1993) Amphetamine and other weak bases act to promote reverse transport of dopamine in ventral midbrain neurons. J Neurochem 60:527ā535. https://doi.org/10.1111/j.1471-4159.1993.tb03181.x
Punja S, Shamseer L, Hartling L et al (2016) Amphetamines for attention deficit hyperactivity disorder (ADHD) in children and adolescents. Cochrane Database Syst Rev 2:CD009996
Castells X, Blanco-Silvente L, Cunill R (2018) Amphetamines for attention deficit hyperactivity disorder (ADHD) in adults. Cochrane Database Syst Rev 8(8):CD007813
Garcia-Olivares J, Baust T, Harris S et al (2017) GĪ²Ī³ subunit activation promotes dopamine efflux through the dopamine transporter. Mol Psychiatry 22:1673ā1679. https://doi.org/10.1038/mp.2017.176
Garcia-Olivares J, Torres-Salazar D et al (2013) Inhibition of dopamine transporter activity by G protein Ī²Ī³ subunits. PLoS One 8(3):e59788. https://doi.org/10.1371/journal.pone.0059788
Gurevich VV, Gurevich EV (2019) GPCR signaling regulation: the role of GRKs and arrestins. Front Pharmacol 10:125. https://doi.org/10.3389/FPHAR.2019.00125
Bungay PM, Newton-Vinson P, Isele W et al (2003) Microdialysis of dopamine interpreted with quantitative model incorporating probe implantation trauma. J Neurochem 86:932ā946. https://doi.org/10.1046/j.1471-4159.2003.01904.x
Yorgason JT, EspaƱa RA, Jones SR (2011) Demon voltammetry and analysis software: analysis of cocaine-induced alterations in dopamine signaling using multiple kinetic measures. J Neurosci Methods 202(2):158ā164. https://doi.org/10.1016/j.jneumeth.2011.03.001
Alonso IP, Pino JA, Kortagere S et al (2021) Dopamine transporter function fluctuates across sleep/wake state: potential impact for addiction. Neuropsychopharmacology 46:699ā708. https://doi.org/10.1038/s41386-020-00879-2
Schelp SA, Brodnik ZD, Rakowski DR et al (2018) Diazepam concurrently increases the frequency and decreases the amplitude of transient dopamine release events in the nucleus accumbens. J Pharmacol Exp Ther 364:145ā155. https://doi.org/10.1124/jpet.117.241802
Bass CE, Grinevich VP, Vance ZB et al (2010) Optogenetic control of striatal dopamine release in rats. J Neurochem 114(5):1344ā1352. https://doi.org/10.1111/j.1471-4159.2010.06850.x
Xiao N, Privman E, Venton BJ (2014) Optogenetic control of serotonin and dopamine release in drosophila larvae. ACS Chem Neurosci 5:666ā673. https://doi.org/10.1021/cn500044b
Sun F, Zeng J, Jing M et al (2018) A genetically encoded fluorescent sensor enables rapid and specific detection of dopamine in flies, fish, and mice. Cell 174:481ā496.e19. https://doi.org/10.1016/j.cell.2018.06.042
Raiteri L, Raiteri M (2000) Synaptosomes still viable after 25 years of superfusion. Neurochem Res 25:1265ā1274. https://doi.org/10.1023/a:1007648229795
Broening HW, William S (1998) Ontogeny of neurotransmitters: monoamines. In: Handbook of developmental neurotoxicology. Academic Press, pp 245ā256. https://doi.org/10.1016/B978-012648860-9.50017-0
Owens WA, Sevak RJ, Galici R et al (2005) Deficits in dopamine clearance and locomotion in hypoinsulinemic rats unmask novel modulation of dopamine transporters by amphetamine. J Neurochem 94:1402ā1410. https://doi.org/10.1111/J.1471-4159.2005.03289.x
Pino JA, Awadallah N, Norris AM, Torres GE (2021) A plate-based assay for the measurement of endogenous monoamine release in acute brain slices. J Vis Exp (174). https://doi.org/10.3791/62127. PMID: 34459801
Koutzoumis DN, Vergara M, Pino J et al (2020) Alterations of the gut microbiota with antibiotics protects dopamine neuron loss and improve motor deficits in a pharmacological rodent model of Parkinsonās disease. Exp Neurol 325:113159. https://doi.org/10.1016/j.expneurol.2019.113159
Torres G, Gainetdinov R, Caron M (2003) Plasma membrane monoamine transporters: structure, regulation and function. Nat Rev Neurosci 4:13ā25. https://doi.org/10.1038/nrn1008
Sulzer D, Chen T, Lau Y et al (1995) Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport. J Neurosci 15:4102ā4108. https://doi.org/10.1523/JNEUROSCI.15-05-04102.1995
Fog JU, Khoshbouei H et al (2006) Calmodulin kinase II interacts with the dopamine transporter C terminus to regulate amphetamine-induced reverse transport. Neuron 51(4):417ā429. https://doi.org/10.1016/j.neuron.2006.06.028. PMID: 16908408
Johnson LA, Guptaroy B, Lund D, Shamban S, Gnegy ME (2005) Regulation of amphetamine-stimulated dopamine efflux by protein kinase C beta. J Biol Chem 280(12):10914ā10919. https://doi.org/10.1074/jbc.M413887200. Epub 2005 Jan 12. PMID: 15647254
Leviel V (2011) Dopamine release mediated by the dopamine transporter, facts and consequences. J Neurochem 118:475ā489
Acknowledgments
We thank the members of the Torres laboratory for their helpful discussions. We also acknowledge the support of the following agencies: Fondecyt Initiation Grant N11191049 (JAP), NIH R01 Grant DA038598 (GET).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
Ā© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Awadallah, N., Pino, J.A., Koutzoumis, D.N., Torres, G.E. (2023). Characterizing Dopamine Transporter Mediated Uptake and Efflux in Brain Slices: A New Approach. In: Fuentealba-Evans, J.A., Henny, P. (eds) Dopaminergic System Function and Dysfunction: Experimental Approaches. Neuromethods, vol 193. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2799-0_4
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2799-0_4
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2798-3
Online ISBN: 978-1-0716-2799-0
eBook Packages: Springer Protocols