JoVE Logo
Faculty Resource Center

Sign In





Representative Results






A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

Published: August 11th, 2021



1Department of Medicine, School of Medicine, Universidad de Atacama, 2Department of Molecular, Cellular, and Biomedical Sciences, City University of New York School of Medicine at City College, 3Department of Pharmacology and Therapeutics, University of Florida College of Medicine
* These authors contributed equally

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

A plethora of neurological and psychiatric diseases are associated with dysregulation or insufficient maintenance of monoamine neurotransmitter (dopamine [DA], serotonin [5-HT], norepinephrine [NE]) homeostasis1,2,3. These conditions include, but are not limited to, depression1,2, schizophrenia2, anxiety2, addiction4, menopause5,6,7, pain

Log in or to access full content. Learn more about your institution’s access to JoVE content here

All experiments, including animal handling and tissue collection, were carried out in accordance with the University of Florida and the City College of New York Institutional Animal Care and Use Committee (IACUC), following the approved protocol 201508873 (UF) and 1071 (CCNY). For reagents and buffer please refer to the Supplementary File.

1. Prepare acute rat brain slices

NOTE: In this experiment adult male rats (250-350 g) were used. However, this s.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This technique describes the use of brain slices to measure the release of endogenous monoamines using HPLC with electrochemical detection based in a 48-well plate with an internal tissue holder. Experimental set up is depicted in Figure 1 and Figure 2. Initially, to ensure tissue viability by the end of the experimentation, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay was performed. Af.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Monoamine release measurements have been performed for years in a number of systems such as heterologous cells, neuronal cultures, brain synaptosomes, ex vivo acute brain slices, and whole animals13,20,41,42,58,64,65,66,67.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This work was supported by grants Fondecyt Initiation Fund N 11191049 to J.A.P. and NIH grant DA038598 to G.E.T.


Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
48 Well plate NA NA Assay
Acetonitrile Fischer Scientific A998-1 Mobile Phase
Calcium Chloride Ahydrous Sigma Aldrich C1016 Modified Artifical Cerebrospinal Fluid OR Efflux Buffer
Clarity Software Anetc
Citric Acid Sigma Aldrich Mobile Phase
D-(+)-Glucose Sigma 1002608421 Dissection Buffer
DMF Sigma Aldrich D4551 MTT Assay
EDTA-Na2 Sigma Aldrich Mobile Phase
GraphPad Software Graphpad Software, Inc Statistical Analysis
Glycerol Sigma Aldrich G5516 Lysis buffer
HEPES Sigma Aldrich H3375 Lysis buffer
HPLC, Decade Amperometric Anetc HPLC, LC-EC system
L-Asrobic Acid Sigma Aldrich A5960 Dissection Buffer
Magnesium Sulfate Sigma 7487-88-9 KH Buffer
Microcentrifuge Filter Units UltraFree Millipore C7554 Assay - 6 to fit in 48 well plate
MTT Thermo Fisher M6494 MTT Assay
Nanosep VWR 29300-606 Assay; protein assay
Octanesulfonic acid Sigma Aldrich V800010 Mobile Phase
Pargyline Clorohydrate Sigma Aldrich P8013 Modified Artifical Cerebrospinal Fluid OR Efflux Buffer
Phosphoric Acid Sigma Aldrich Mobile Phase
Potassium Chloride Sigma 12636 KH Buffer
Potassium Phosphate Monobasic Sigma 1001655559 KH Buffer
Precisonary VF-21-0Z Precissonary Compresstome
Protease Inhibitor Cocktail Sigma Aldrich P2714 Lysis buffer.
Sodium Bicarbonate Sigma S5761 Dissection Buffer
Sodium Bicarbonate Sigma Aldrich S5761 Dissection Buffer
Sodium Chloride Sigma S3014 KH Buffer
Sodium Dodecyl Sulfate Sigma Aldrich L3771 Lysis buffer
Triton X-100 Sigma Aldrich T8787 MTT Assay / Lysis buffer

  1. Jesulola, E., Micalos, P., Baguley, I. J. Understanding the pathophysiology of depression: From monoamines to the neurogenesis hypothesis model - are we there yet. Behavioural Brain Research. 341, 79-90 (2018).
  2. Krystal, J. H., D'Souza, D. C., Sanacora, G., Goddard, A. W., Charney, D. S. Current perspectives on the pathophysiology of schizophrenia, depression, and anxiety disorders. Medical Clinics of North America. 85 (3), 559-577 (2001).
  3. Barone, P. Neurotransmission in Parkinson's disease: beyond dopamine. European Journal of Neurology. 17 (3), 364-376 (2010).
  4. Howell, L. L., Kimmel, H. L. Monoamine transporters and psychostimulant addiction. Biochemical Pharmacology. 75 (1), 196-217 (2008).
  5. Kirshner, Z. Z., et al. Impact of estrogen receptor agonists and model of menopause on enzymes involved in brain metabolism, acetyl-CoA production and cholinergic function. Life Sciences. 256, 117975 (2020).
  6. Long, T., et al. Comparison of transitional vs surgical menopause on monoamine and amino acid levels in the rat brain. Molecular and Cellular Endocrinology. 476, 139-147 (2018).
  7. Long, T., et al. Estradiol and selective estrogen receptor agonists differentially affect brain monoamines and amino acids levels in transitional and surgical menopausal rat models. Molecular and Cellular Endocrinology. 496, 110533 (2019).
  8. Burke, N. N., et al. Enhanced nociceptive responding in two rat models of depression is associated with alterations in monoamine levels in discrete brain regions. Neuroscience. 171 (4), 1300-1313 (2010).
  9. Lane, J. D., Aprison, M. H. Calciumm-dependent release of endogenous serotonin, dopamine and norepinephrine from nerve endings. Life Sciences. 20 (4), 665-671 (1977).
  10. Ramamoorthy, S., Shippenberg, T. S., Jayanthi, L. D. Regulation of monoamine transporters: Role of transporter phosphorylation. Pharmacology and Therapeutics. 129 (2), 220-238 (2011).
  11. Torres, G. E., Gainetdinov, R. R., Caron, M. G. Plasma membrane monoamine transporters: structure, regulation and function. Nature Reviews. Neuroscience. 4 (1), 13-25 (2003).
  12. Hilber, B., et al. Serotonin-transporter mediated efflux: A pharmacological analysis of amphetamines and non-amphetamines. Neuropharmacology. 49 (6), 811-819 (2005).
  13. Mauna, J. C., et al. G protein βγ subunits play a critical role in the actions of amphetamine. Translational Psychiatry. 9 (1), 81 (2019).
  14. Sitte, H. H., Freissmuth, M. Amphetamines, new psychoactive drugs and the monoamine transporter cycle. Trends in Pharmacological Sciences. 36 (1), 41-50 (2015).
  15. Johnson, L. A., Guptaroy, B., Lund, D., Shamban, S., Gnegy, M. E. Regulation of amphetamine-stimulated dopamine efflux by protein kinase C β. Journal of Biological Chemistry. 280 (12), 10914-10919 (2005).
  16. Kahlig, K. M., et al. Amphetamine induces dopamine efflux through a dopamine transporter channel. Proceedings of the National Academy of Sciences of the United States of America. 102 (9), 3495-3500 (2005).
  17. Kantor, L., Hewlett, G. H. K., Gnegy, M. E. Enhanced amphetamine- and K+ -mediated dopamine release in rat striatum after repeated amphetamine: differential requirements for Ca 2+ - and calmodulin-dependent phosphorylation and synaptic vesicles. The Journal of Neuroscience. 19 (10), 3801-3808 (2018).
  18. Brodnik, Z. D., et al. Susceptibility to traumatic stress sensitizes the dopaminergic response to cocaine and increases motivation for cocaine. Neuropharmacology. 125, 295-307 (2017).
  19. Henke, A., et al. Toward serotonin fluorescent false neurotransmitters: development of fluorescent dual serotonin and vesicular monoamine transporter substrates for visualizing serotonin neurons. ACS Chemical Neuroscience. 9 (5), 925-934 (2018).
  20. Garcia-Olivares, J., et al. Gβγ subunit activation promotes dopamine efflux through the dopamine transporter. Molecular Psychiatry. 22 (12), 1673-1679 (2017).
  21. Xiao, N., Privman, E., Venton, B. J. Optogenetic control of serotonin and dopamine release in Drosophila larvae. ACS Chemical Neuroscience. 5 (8), 666-673 (2014).
  22. Bass, C. E., et al. Optogenetic control of striatal dopamine release in rats. Journal of Neurochemistry. 114 (5), 1344-1352 (2010).
  23. Stamford, J. A. Fast cyclic voltammetry: measuring transmitter release in "real time". Journal of Neuroscience Methods. 34 (1-3), 67-72 (1990).
  24. Brodnik, Z. D., Ferris, M. J., Jones, S. R., España, R. A. Reinforcing doses of intravenous cocaine produce only modest dopamine uptake inhibition. ACS Chemical Neuroscience. 8 (2), 281-289 (2017).
  25. Brodnik, Z. D., España, R. A. Dopamine uptake dynamics are preserved under isoflurane anesthesia. Neuroscience Letters. 606, 129-134 (2015).
  26. Ferris, M. J., Calipari, E. S., Yorgason, J. T., Jones, S. R. Examining the complex regulation and drug-induced plasticity of dopamine release and uptake using voltammetry in brain slices. ACS Chemical Neuroscience. 4 (5), 693-703 (2013).
  27. Siciliano, C. A., Calipari, E. S., Ferris, M. J., Jones, S. R. Biphasic mechanisms of amphetamine action at the dopamine terminal. The Journal of Neuroscience The Official Journal of the Society for Neuroscience. 34 (16), 5575-5582 (2014).
  28. Rice, M. E., et al. Direct monitoring of dopamine and 5-HT release in substantia nigra and ventral tegmental area in vitro. Experimental Brain Research. 100 (3), 395-406 (1994).
  29. Bunin, M. A., Prioleau, C., Mailman, R. B., Wightman, R. M. Release and uptake rates of 5-hydroxytryptamine in the dorsal raphe and substantia nigra reticulata of the rat brain. Journal of Neurochemistry. 70 (3), 1077-1087 (1998).
  30. Park, J., Takmakov, P., Wightman, R. M. In vivo comparison of norepinephrine and dopamine release in rat brain by simultaneous measurements with fast-scan cyclic voltammetry. Journal of Neurochemistry. 119 (5), 932-944 (2011).
  31. Park, J., Bhimani, R. V., Bass, C. E. In vivo electrochemical measurements of norepinephrine in the brain: current status and remaining challenges. Journal of the Electrochemical Society. 165 (12), 3051-3056 (2018).
  32. Butcher, S. P., Fairbrother, I. S., Kelly, J. S., Arbuthnott, G. W. Amphetamine-induced dopamine release in the rat striatum: an in vivo microdialysis study. Journal of Neurochemistry. 50 (2), 346-355 (1988).
  33. Garcia-Olivares, J., et al. Inhibition of dopamine transporter activity by G protein βγ subunits. PLoS One. 8 (3), 1-9 (2013).
  34. Carneiro, A. M. D., Blakely, R. D. Serotonin-, protein kinase C-, and Hic-5-associated redistribution of the platelet serotonin transporter. Journal of Biological Chemistry. 281 (34), 24769-24780 (2006).
  35. Rajamanickam, J., et al. Akt-mediated regulation of antidepressant-sensitive serotonin transporter function, cell-surface expression and phosphorylation. The Biochemical Journal. 468 (1), 177-190 (2015).
  36. Egaña, L. A., et al. Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3. The Journal of Neuroscience The Official Journal of the Society for Neuroscience. 29 (14), 4592-4604 (2009).
  37. Guptaroy, B., Fraser, R., Desai, A., Zhang, M., Gnegy, M. E. Site-directed mutations near transmembrane domain 1 alter conformation and function of norepinephrine and dopamine transporters. Molecular Pharmacology. 79 (3), 520-532 (2011).
  38. Ordway, G. A., et al. Norepinephrine transporter function and desipramine: Residual drug effects versus short-term regulation. Journal of Neuroscience Methods. 143 (2), 217-225 (2005).
  39. Steinkellner, T., et al. Amphetamine action at the cocaine- and antidepressant-sensitive serotonin transporter is modulated by CaMKII. Journal of Neuroscience. 35 (21), 8258-8271 (2015).
  40. Guptaroy, B., et al. A juxtamembrane mutation in the N terminus of the dopamine transporter induces preference for an inward-facing conformation. Molecular Pharmacology. 75 (3), 514-524 (2009).
  41. Carpenter, C., et al. Direct and systemic administration of a CNS-permeant tamoxifen analog reduces amphetamine-induced dopamine release and reinforcing effects. Neuropsychopharmacology. 42 (10), 1940-1949 (2017).
  42. Aquino-Miranda, G., Escamilla-Sánchez, J., González-Pantoja, R., Bueno-Nava, A., Arias-Montaño, J. -. A. Histamine H3 receptor activation inhibits dopamine synthesis but not release or uptake in rat nucleus accumbens. Neuropharmacology. 106, 91-101 (2016).
  43. Reddy, I. A., et al. Glucagon-like peptide 1 receptor activation regulates cocaine actions and dopamine homeostasis in the lateral septum by decreasing arachidonic acid levels. Translational Psychiatry. 6 (5), 809 (2016).
  44. Koutzoumis, D. N., et al. Alterations of the gut microbiota with antibiotics protects dopamine neuron loss and improve motor deficits in a pharmacological rodent model of Parkinson's disease. Experimental Neurology. 325, 113159 (2020).
  45. Herdon, H., Strupish, J., Nahorski, S. R. Differences between the release of radiolabelled and endogenous dopamine from superfused rat brain slices: Effects of depolarizing stimuli, amphetamine and synthesis inhibition. Brain Research. 348 (2), 309-320 (1985).
  46. Thongsaard, W., Kendall, D. A., Bennett, G. W., Marsden, C. A. A simple method for measuring dopamine release from rat brain slices. Journal of Pharmacological and Toxicological Methods. 37 (3), 143-148 (1997).
  47. Dorris, D. M., Hauser, C. A., Minnehan, C. E., Meitzen, J. An aerator for brain slice experiments in individual cell culture plate wells. Journal of Neuroscience Methods. 238, 1-10 (2014).
  48. Humpel, C. Organotypic brain slice cultures: a review. Neuroscience. 305, 86-98 (2015).
  49. Papouin, T., Haydon, P. Obtaining acute brain slices. BIO-PROTOCOL. 8 (2), 477-491 (2018).
  50. Collingridge, G. L. The brain slice preparation: a tribute to the pioneer Henry McIlwain. Journal of Neuroscience Methods. 59 (1), 5-9 (1995).
  51. Yamamoto, C., McIlwain, H. Electrical activities in thin sections from the mammalian brain maintained in chemically-defined media in vitro. Journal of Neurochemistry. 13 (12), 1333-1343 (1966).
  52. Buskila, Y., et al. Extending the viability of acute brain slices. Scientific Reports. 4, 4-10 (2014).
  53. Kako, H., Fukumoto, S., Kobayashi, Y., Yokogoshi, H. Effects of direct exposure of green odour components on dopamine release from rat brain striatal slices and PC12 cells. Brain Research Bulletin. 75 (5), 706-712 (2008).
  54. McBride, W. J., Murphy, J. M., Lumeng, L., Li, T. -. K. Effects of ethanol on monoamine and amino acid release from cerebral cortical slices of the alcohol-preferring P line of rats. Alcoholism: Clinical and Experimental Research. 10 (2), 205-208 (1986).
  55. Chen, J. C., Turiak, G., Galler, J., Volicer, L. Effect of prenatal malnutrition on release of monoamines from hippocampal slices. Life Sciences. 57 (16), 1467-1475 (1995).
  56. Becker, J. B., Castañeda, E., Robinson, T. E., Beer, M. E. A simple in vitro technique to measure the release of endogenous dopamine and dihydroxyphenylacetic acid from striatal tissue using high performance liquid chromatography with electrochemical detection. Journal of Neuroscience Methods. 11 (1), 19-28 (1984).
  57. Paxinos, G., Watson, C. . The Rat Brain in Stereotaxic Coordinates. , (2007).
  58. Dailey, J. W., Reith, M. E. A., Steidley, K. R., Milbrandt, J. C., Jobe, P. C. Carbamazepine-induced release of serotonin from rat hippocampus in vitro. Epilepsia. 39 (10), 1054-1063 (1998).
  59. Buskila, Y., et al. Extending the viability of acute brain slices. Scientific Reports. 4, 5309 (2014).
  60. Mewes, A., Franke, H., Singer, D. Organotypic brain slice cultures of adult transgenic P301S mice-A model for tauopathy studies. PLoS One. 7 (9), (2012).
  61. Rönicke, R., et al. AB mediated diminution of MTT reduction - An artefact of single cell culture. PLoS One. 3 (9), (2008).
  62. Ihalainen, J. A., Riekkinen, P., Feenstra, M. G. P. Comparison of dopamine and noradrenaline release in mouse prefrontal cortex, striatum and hippocampus using microdialysis. Neuroscience Letters. 277 (2), 71-74 (1999).
  63. Richards, D. A., Obrenovitch, T. P., Symon, L., Curzon, G. Extracellular dopamine and serotonin in the rat striatum during transient ischaemia of different severities: a microdialysis study. Journal of Neurochemistry. 60 (1), 128-136 (1993).
  64. Fog, J. U., et al. Calmodulin kinase II interacts with the dopamine transporter C terminus to regulate amphetamine-induced reverse transport. Neuron. 51 (4), 417-429 (2006).
  65. Balázsa, T., Bíró, J., Gullai, N., Ledent, C., Sperlágh, B. CB1-cannabinoid receptors are involved in the modulation of non-synaptic [3H]serotonin release from the rat hippocampus. Neurochemistry International. 52 (1), 95-102 (2008).
  66. Schechter, L. E. The potassium channel blockers 4-aminopyridine and tetraethylammonium increase the spontaneous basal release of [3H]5-hydroxytryptamine in rat hippocampal slices. The Journal of Pharmacology and Experimental Therapeutics. 282 (1), 262-270 (1997).
  67. Boudanova, E., Navaroli, D. M., Stevens, Z., Melikian, H. E. Dopamine transporter endocytic determinants: carboxy terminal residues critical for basal and PKC-stimulated internalization. Molecular and Cellular Neuroscience. 39 (2), 211-217 (2008).
  68. Bowyer, J. F., et al. Interactions of MK-801 with glutamate-, glutamine- and methamphetamine-evoked release of [3H]dopamine from striatal slices. The Journal of Pharmacology and Experimental Therapeutics. 257 (1), 262-270 (1991).
  69. Perszyk, R. E., et al. GluN2D-containing N-methyl-D-aspartate receptors mediate synaptic transmission in hippocampal interneurons and regulate interneuron activity. Molecular Pharmacology. 90 (6), 689-702 (2016).
  70. Jones, S. R., et al. Profound neuronal plasticity in response to inactivation of the dopamine transporter. Proceedings of the National Academy of Sciences of the United States of America. 95 (7), 4029-4034 (1998).
  71. Jedema, H. P., Narendran, R., Bradberry, C. W. Amphetamine-induced release of dopamine in primate prefrontal cortex and striatum: striking differences in magnitude and timecourse. Journal of Neurochemistry. 130, 490-497 (2014).
  72. Buchmayer, F., et al. Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate. Proceedings of the National Academy of Sciences. 110 (28), 11642-11647 (2013).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved