A Plate-Based Assay to Measure Monoamine Release from Brain Slices on Test Drug Exposure

Published: April 30, 2023

Abstract

Source: Pino, J. A. et al. A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices. J. Vis. Exp. (2021)

In this video, we demonstrate an assay that investigates monoamine release mechanisms from the neurons of metabolically active ex vivo rat brain slices, when treated with test drugs. The assay demonstrates that neurostimulant drugs induce monoamine release via the cellular monoamine transporters, whereas high KCl concentration induces synaptic vesicle exocytosis-mediated vesicular monoamine release.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Ex-vivo endogenous monoamine release from brain slices or punches NOTE: The device used for this section consists of a 48-well plate and a tissue holder made of six microcentrifuge filter units without the inset filters connected to a carbogen line (Figure 1). To make the holder, use a stu…

Representative Results

Figure 1: Experimental setup for efflux experiment. (A) The efflux chamber consists of a 48-well tissue culture plate and a tissue holder tray connected to the carbogen line. (B) A diagram showing the experimental design for the endogenous monoamine efflux experiment in which the tissue activation (B1), pre-incubation with/without monoamine transporter inhibitors…

開示

The authors have nothing to disclose.

Materials

48-well plate NA NA Assay
Calcium Chloride Anhydrous Sigma Aldrich C1016 Modified Artifical Cerebrospinal Fluid OR Efflux Buffer
D-(+)-Glucose Sigma 1002608421 Dissection Buffer
L-Ascorbic 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
Pargyline Clorohydrate Sigma Aldrich P8013 Modified Artifical Cerebrospinal Fluid OR Efflux Buffer
Potassium Chloride Sigma 12636 KH Buffer
Potassium Phosphate Monobasic Sigma 1001655559 KH Buffer
Sodium Bicarbonate Sigma S5761 Dissection Buffer
Sodium Bicarbonate Sigma Aldrich S5761 Dissection Buffer
Sodium Chloride Sigma S3014 KH Buffer

タグ

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記事を引用
A Plate-Based Assay to Measure Monoamine Release from Brain Slices on Test Drug Exposure. J. Vis. Exp. (Pending Publication), e21279, doi: (2023).

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