This study investigates the role of monoamine neurotransmitters in neuronal function and the effects of a test drug on their release. Using rat brain tissue slices, the research outlines a method to assess monoamine concentration changes in response to pharmacological agents.
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.
To induce monoamine release, transfer the tissue samples to each well of a custom-made 48-well efflux chamber, containing 0.5 to 1 milliliter of efflux buffer per well with constant, gentle bubbling, and allow the samples to recover for 30 to 50 minutes at 37 degrees Celsius.
At the end of the equilibration period, move the tissue holder with brain tissue, to wells containing 500 microliters of oxygenated efflux buffer with or without pharmacological agent, tapping the holder on the edge of the well to prevent the minimal transfer of buffer between wells, for a 20-minute incubation at 37 degrees Celsius.
At the end of the incubation, move the holder to a new set of wells containing 500 microliters of efflux buffer with or without the drug of interest, and return the plate to the cell culture incubator, for an additional 20 minutes.
During the second incubation, transfer the solution from the wells from the first incubation into microcentrifuge tubes containing 50 microliters of 1 N perchloric or phosphoric acid on ice, and label the tubes #1.
At the end of the second incubation, move the tissue holder to empty wells with the plate on ice, and transfer the supernatants from the second incubation to new microcentrifuge tubes containing 50 microliters of 1 N perchloric or phosphoric acid, on ice. Label these tubes #2.
When all of the supernatants have been transferred, add 1 milliliter of ice-cold dissection buffer to each well of tissue, and use small tweezers to transfer each tissue sample to a new microcentrifuge tube. Remove the buffer from the sample tubes, and store the tissues at -80 degrees Celsius. Then, transfer the collected incubation solutions into microcentrifuge filter tubes for centrifugation, and place the filtrate on ice, until HPLC analysis.
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