Method Article

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

DOI:

10.3791/56093

⸱

September 21st, 2017

 ,  ,  ,  , 
1Molecular Neuropharmacology and Genetics Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2Laboratory of Neuropsychiatry, Psychiatric Center Copenhagen and Department of Neuroscience and Pharmacology, University of Copenhagen

In This Article

Summary

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Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Abstract

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Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson's disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.

Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.

DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.

Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Introduction

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Dopamine (DA) is a modulatory neurotransmitter critical for motor behaviour, reward and cognitive function1,7,8,9. Imbalances in DA homeostasis are implicated in several neuropsychiatric diseases such as attention-deficit hyperactivity disorder, drug addiction, depression and Parkinson's disease1. DA is released from the presynaptic neuron into the synaptic cleft, where it binds to and activates receptors on the pre- and postsynaptic membrane, thereby further conveying the signal. The level of DA in the synapse af....

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Protocol

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The guidelines of the Danish Animal Experimentation Inspectorate (permission number: 2017-15-0201-01160) was followed and experiments performed in a fully AAALAC accredited facility under the supervision of a local animal welfare committee.

1. Synaptosomal Dopamine Uptake (Method 1)

NOTE: This protocol is for parallel assessment of two brains, but can be successfully used to perform synaptosomal DA uptake experiments with four brains in parallel.

  1. Preparations
    1. Label 48 1.5 mL micro-centrifuge tubes per Table 1 (24 for each brain). Use different colours for ....

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Results

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Current DA uptake protocol (Figure 1) includes all steps necessary to assess the functionality of DAT in synaptosomes from mice. Our representative data of the DA uptake method (Figure 2) depicts a saturation curve with unadjusted data (Figure 2B) and adjusted data (Figure 2A). The saturation curve shows uptake from wild type mice. Usually one would make DA uptake for co.......

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Discussion

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This manuscript describes useful experimental protocols to delineate DA homeostasis in any mouse model of choice. We provide detailed protocols for measuring levels of DA in brain tissue from mice using HPLC and synaptosomal DA uptake to assess functional DA transport through DAT. The procedures, protocols and limits for the HPLC experiment and synaptosomal DA uptake assay will be elaborated below.

The synaptosomal uptake protocol can provide useful insight to the functionality of DAT. Combine.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This work was supported by the UCPH 2016 Program of Excellence (U.G., A.R., K.J.), the Lundbeck Foundation (M.R.) the Lundbeck Foundation Center for Biomembranes in Nanomedicine (U.G.), the National Institute of Health Grants P01 DA 12408 (U.G.), the Danish Council for independent Research - Medical Sciences (U.G.).

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
COMT inhibitorSigma Aldrich, GermanyRO-41-0960For synaptosomal DA uptake protocol
[3H]-DopaminePerkin-Elmer Life Sciences, Boston, MA, USANET67-3001MCFor synaptosomal DA uptake protocol
Glass microfiber filtersGF/C Whatman, GE Healthcare Life Sciences, Buckinghamshire1822-024For synaptosomal DA uptake protocol
HiSafe Scintillation fluidPerkin Elmer1200-437For synaptosomal DA uptake protocol
MicroBeta2Perkin ElmerFor synaptosomal DA uptake protocol
BCA Protein Assay kitThermo Scientific Pierce23225For synaptosomal DA uptake protocol
HEPESSigma Life ScienceH3375For synaptosomal DA uptake protocol
SucroseSigma Life ScienceS7903For synaptosomal DA uptake protocol
NaClSigma Life ScienceS3014For synaptosomal DA uptake protocol
KClSigma Life ScienceP9541For synaptosomal DA uptake protocol
CaCl2Merck KGaA10043-52-4For synaptosomal DA uptake protocol
MgSO4Sigma Life Science63065For synaptosomal DA uptake protocol
Ascorbic AcidSigma Life ScienceA0278For synaptosomal DA uptake protocol
D-GlucoseSigma Life ScienceG7021For synaptosomal DA uptake protocol
PargylineSigma AldrichP-8013For synaptosomal DA uptake protocol
DesipramineSigma AldrichD3900For synaptosomal DA uptake protocol
DopamineSigma Life ScienceH8502For synaptosomal DA uptake protocol
CocaineSigma Life ScienceC5776For synaptosomal DA uptake protocol
Brain matrixASI instrumentsRBM2000CFor synaptosomal DA uptake protocol
Cafano mechanical teflon disrupterBuch & HolmDiscontinuedFor synaptosomal DA uptake protocol (homogenization)
Antec Decade (Amperometric detector)Antec, Leiden, The NetherlandsDiscontinued: new model DECADE Elite / Lite™ Electrochemical Detector type 175 and 176For HPLC protocol
Avantec 0.22 μm glass filterFrisenette ApS, Denmark13CP020ASFor HPLC protocol
Column: Prodigy 3 μ ODS-3 C18Phenomenex, YMC Europe, Chermbeck, GermanyPart Number:00A-3300-E0For HPLC protocol
LC solution softwareShimadzuLabSolutions Series WorkstationFor HPLC protocol
Perchlor acid 0.1MFluka Analytical35418-500mlFor HPLC protocol (Tissue preparation)
EDTASigmaE5134-50gFor HPLC protocol
NatriumdihydrogenphospharBie&Berntsen1.06346 1000gFor HPLC protocol
Sodium 1-octanesulfonate monohydrateAldrich74885 -10gFor HPLC protocol
Acetonitrile, isocratic HPLC gradeScharlauAC03402500For HPLC protocol
Filtre 0.22umFrisenette ApS, DenmarkAvantec 13CP020ASFor HPLC protocol (Tissue preparation)
ortho-Phosphoric acid 85%Merck1.00563. 1000mlFor HPLC protocol
ElectrodeAntec, Leiden, The NetherlandsAN1161300For HPLC protocol (see manual online)
Detector program on DECADE II electrochemical detectorAntec, Leiden, The NetherlandsLite™ Electrochemical Detector type 175 and 176For HPLC protocol

References

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  1. Tritsch, N. X., Sabatini, B. L. Dopaminergic modulation of synaptic transmission in cortex and striatum. Neuron. 76, 33-50 (2012).
  2. Cartier, E. A., et al. A biochemical and functional protein complex involving dopamine synthesis and tr....

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Tags

Dopamine HomeostasisDopamine TransporterDopamine LevelsStriatal TissueDopamine Uptake AssayBrain Tissue AnalysisDopamine Transporter FunctionDopamine Transporter Kinetics

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