A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

This technique is an adaptation of commonly used methods for examining monoamine release, allowing the measurement of endogenous release rather than preloaded radiolabeled monoamines under multiple pharmacological conditions at one time. The main advantages to this technique are that it is fairly cost-effective, multiple pharmacological conditions may be examined at once, and we can study endogenous monoamine release with the ability to distinguish between vesicular and transporter mediated release. To prepare brain slices from adult 250 to 350 gram male rats after harvest, immediately placed the brain and ice cold oxygenated dissection buffer, and acquire 300 micron coronal brain sections from each region of interest.

For further dissection of the brain slices, carefully move the tissues to glass slides and use a rat brain atlas to identify the dorsal striatum based on its dark striated structure, and to identify the hippocampus based on its proximity to the cortex and its unique spiral structure. Separate the right and left hemispheres for their use as control and experimental slices. The dorsal striatum can be further dissected into two millimeter punches.

Then use a plastic transfer pipette with a modified tip to transfer the samples into small containers immersed in oxygenated ice cold dissection buffer with oxygen bubbling. To induce monoamine release, transfer the tissue samples to each well of a custom made 48 well efflux chamber containing 0.5 to one 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 one normal perchloric or phosphoric acid on ice, and label the tubes number one"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 one normal perchloric or phosphoric acid on ice. Label these tubes number two"When all of the supernatants have been transferred, add one 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 minus 80 degrees Celsius, then transfer the collected incubation solutions into microcentrifuge filter tubes for centrifugation and place the filtrate on ice until HPLC analysis. Set the potential of the ECD and flow rate to the manufacturer's recommendation for detecting monoamines. Load and appropriate aliquot of each sample, including neuro-transmitter standards into the HPLC for auto injection and detection and allow the instrument to complete the analysis.

Then use the HPLC analysis software to acquire and analyze the chromatograph data using a standard curve composed of each monoamine to obtain the area under the curve based on the manufacturer's guidelines. After six hours of functional analysis, tissue samples remain viable, while samples incubated with 1%Triton X-100 demonstrate a significant reduction in MTT transformation. Acute 20 minute treatment of hippocampal and prefrontal cortex brain sections with amphetamine induces a significant increase in the extracellular level of each monoamine.

When the samples are pretreated with fluoxetine, a selective serotonin re-uptake inhibitor, the amphetamine induced increase in extracellular serotonin within the hippocampus and the prefrontal cortex does not occur, while the increase in extracellular dopamine and norepinephrine production is not affected. In addition, acute 20 minute treatment of dorsal striatum punches with amphetamine induces a 35 fold increase in extracellular dopamine level expression, while dorsal striatum punch treatment with cocaine, a monoamine transporter blocker, results in a significant inhibition of amphetamine induced extracellular dopamine. Increasing the concentration of extracellular potassium chloride to evoke membrane depolarization is sufficient to induce the exocytotic release of monoamines compared to untreated control conditions, and neither fluoxetine nor cocaine treatment blocks this deep polarization induced increase.

It is imperative to have clean cut sections, a precise localization of punches, and to maintain consistent oxygenation throughout the experiment. The brain sections may be used post experimentation for further biochemical processing and analysis, such as western blotting and histology.