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

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…

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