A Plate-Based Assay to Measure Monoamine Release from Brain Slices on Test Drug Exposure
Transcription
Monoamine neurotransmitters, endogenous chemical messengers, relay signals across the synapse, the point of contact between neurons. Tightly regulated monoamine neurotransmitter system is essential for normal neuronal function.
To screen the effect of a test drug on monoamine release ex vivo, obtain rat brain tissue slices containing the region of interest in a chilled oxygenated buffer, maintaining tissue viability.
Transfer the tissue slices to wells of a multi-well plate, with porous filter inserts connected to a gaseous mixture supply and comprising oxygenated artificial cerebrospinal fluid, aCSF, supplemented with monoamine oxidase inhibitors. Incubate.
The inhibitors diffuse into the cells and inactivate monoamine oxidase enzymes, preventing degradation of monoamines and increasing their concentration available for storage and release.
Transfer the tissue-containing inserts to wells comprising oxygenated aCSF, and the test drug. Incubate. The drug may stimulate the monoamine neurotransmitter system, increasing the monoamine concentration in the extraneuronal space, leading to their release from the brain tissue into solution.
The released monoamines pass through the porous inserts into the wells, separating from the tissue.
Post-incubation, remove the tissue slice-containing inserts. Collect the monoamine solution in a tube comprising a solvent compatible with high-performance liquid chromatography, HPLC. Transfer the tube contents into pre-assembled filter units. Centrifuge, removing major contaminants.
Load the obtained filtrate into the HPLC column and perform a chromatography run to determine monoamine concentration, indicative of the test drug effect on monoamine release.
