Measuring Dopamine Uptake in Mouse Brain Synaptosomes Using Radiolabeled Dopamine

In This Article

Abstract

Source: Jensen, K. L., et al., Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake. J. Vis. Exp. (2017).

This video demonstrates the assessment of dopamine uptake in synaptosomes harvested from mouse brains. It outlines steps for preparing synaptosomes for dopamine uptake via dopamine transporters, treating them with varying concentrations of radiolabeled dopamine, isolating the synaptosomes, and measuring radioactivity to quantify dopamine uptake.

Protocol

1. Synaptosomal Dopamine Uptake

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

Preparations
1. Label 48 1.5 mL micro-centrifuge tubes per Table 1 (24 for each brain). Use different colors for the tubes belonging to each brain to prevent confusion between samples.
2. Label 51 scintillation tubes #1-51.
3. Place phosphate buffered saline (PBS) on ice. This will be used to keep brains chilled.

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Results

Brain dissection process diagram; homogenization, centrifugation; DA uptake data analysis graph.
Figure 1: Schematic workflow depicting the different steps in the synaptosomal DA uptake experiment protocol. The mouse is sacrificed by cervical dislocation, followed by brain dissection and placement in a brain matrix. A 3 mm thick coronal slice is dissec.......