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