Name: a plate-based assay for the measurement of endogenous monoamine release in acute brain slices
Uploaded: 2021-08-11T13:00:00
Description: Watch this Scientific Journal Video about A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices at JoVE.com

This work was supported by grants Fondecyt Initiation Fund N 11191049 to J.A.P. and NIH grant DA038598 to G.E.T.

All experiments, including animal handling and tissue collection, were carried out in accordance with the University of Florida and the City College of New York Institutional Animal Care and Use Committee (IACUC), following the approved protocol 201508873 (UF) and 1071 (CCNY). For reagents and buffer please refer to the Supplementary File.
1. Prepare acute rat brain slices
NOTE: In this experiment adult male rats (250-350 g) were used. However, this s...

Monoamine release measurements have been performed for years in a number of systems such as heterologous cells, neuronal cultures, brain synaptosomes, ex vivo acute brain slices, and whole animals13,20,41,42,58,64,65,66,67...

A plethora of neurological and psychiatric diseases are associated with dysregulation or insufficient maintenance of monoamine neurotransmitter (dopamine [DA], serotonin [5-HT], norepinephrine [NE]) homeostasis1,2,3. These conditions include, but are not limited to, depression1,2, schizophrenia2, anxiety2, addiction4, menopause5,6,7, pain8, and Parkinson&#39;s disease3. For instance, several rat models of menopause have shown that the dysregulation or reduction of monoamines within the hippocampus, prefrontal cortex, and striatum may be associated with both depression and cognitive decline, which is seen in women experiencing menopause. The dysregulation of monoamines in these models have been extensively examined using HPLC-ECD, although the studies did not discriminate between measured neurotransmitter content versus neurotransmitter release5,6,7. Monoamines are classically released into the extracellular space through Ca2+-dependent vesicular release9, and are recycled back through their respective plasma membrane re-uptake system (dopamine transporter, DAT; serotonin transporter, SERT; norepinephrine transporter, NET)10,11. Conversely, data suggests that these transporters are able to release or efflux monoamines, since drugs of abuse such as amphetamine (AMPH) and 3,4-Methylenedioxymethamphetamine (MDMA) are known to release DA and 5-HT, respectively through their transporter systems12,13,14,15,16,17. Thus, a proper mechanistic understanding of monoamine release dynamics is crucial for developing specific and targeted pharmacotherapies.
A wide range of techniques have been employed to study monoamine release such as Fast Scan Cyclic Voltammetry (FSCV)18, in vivo&#160;microdialysis13, imaging19, preincubation with radiolabeled monoamines20, optogenetics, and more recently, genetically encoded fluorescent sensors and photometry21,22. FSCV and in vivo microdialysis are the primary techniques used for studying monoamine release. FSCV is used to study the stimulated exocytotic release of, primarily, DA in acute brain slices and in vivo23. Because FSCV uses electrodes to stimulate or evoke release, the primary source of neurotransmitter release is Ca2+-dependent vesicular release18,24,25,26,27,28,29,30,31. In vivo&#160;microdialysis coupled with HPLC measures changes in extracellular neurotransmitter levels using a probe placed in a brain area of interest13,32. Similar to FSCV, a major limitation to in vivo&#160;microdialysis is the difficulty in determining the source of neurotransmitter release: Ca2+ dependent vesicular release or transporter dependent. Noteworthy, both methods allow for the direct measurement of monoamine release. Through the recent advancement of optogenetics, research demonstrates detection of 5-HT and DA release in a short timespan with exquisite cell-type specificity21,22. However, these strategies require complex and costly techniques and equipment, and indirectly measure monoamine release, specifically through monoamine binding to receptors. Further, radiolabeled monoamines are also used for studying monoamine dynamics. Radiolabeled monoamines may be preloaded into various model systems such as heterologous cells overexpressing each monoamine transporter20,33,34,35,36,37,38,39,40, primary neurons20, synaptosomes33,39,41,42, and acute brain slices43,44. However, radioactivity poses potential harm to the experimenter, and the tritium-labeled analytes may not faithfully recapitulate endogenous monoamine dynamics45,46. Superfusion systems combined with off-line detection methods such as HPLC-ECD have allowed for the detection of monoamines from multiple tissue sources. Here, this protocol provides as an optimized and low-cost, simple, and precise method using acute brain slices to directly measure endogenous basal and stimulated monoamine release.
Acute brain slices allow for testing mechanistic hypotheses, primarily as they preserve the in vivo anatomical microenvironment, and maintain intact synapses47,48,49,50,51,52. In a few studies, acute brain slices or chopped brain tissue have been used in conjunction with a superfusion technique using KCl to stimulate Ca2+ mediated release53,54,55,56. Superfusion systems have been critical to advance the field&#39;s understanding of neurotransmitter release mechanisms, including monoamines. However, these systems are relatively expensive, and the number of chambers available for tissue analysis ranges from 4-12. In comparison, the method presented here is inexpensive, allows the measurement of 48 tissue samples, and may be refined to use up to 96 tissue samples. Each well within the 48-well plate contains tissue holders that use filters to separate the released product from the tissue, and released monoamines are then collected and analyzed by HPLC-ECD. Importantly, this method allows for the simultaneous measurement of 5-HT, DA, and NE release from different brain areas such as the prefrontal cortex, the hippocampus, and the dorsal striatum after treatment with pharmacological agents that modulate monoamine release. Thus, the experimenter can answer multiple questions using an inexpensive multi-well system that increases the number of samples tested and thereby reducing the number of animals used.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>48 Well plate</td>
			<td>NA</td>
			<td>NA</td>
			<td>Assay</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fischer Scientific</td>
			<td>A998-1</td>
			<td>Mobile Phase</td>
		</tr>
		<tr>
			<td>Calcium Chloride Ahydrous</td>
			<td>Sigma Aldrich</td>
			<td>C1016</td>
			<td>Modified Artifical Cerebrospinal Fluid OR Efflux Buffer</td>
		</tr>
		<tr>
			<td>Clarity Software</td>
			<td>Anetc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>Mobile Phase</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>1002608421</td>
			<td>Dissection Buffer</td>
		</tr>
		<tr>
			<td>DMF</td>
			<td>Sigma Aldrich</td>
			<td>D4551</td>
			<td>MTT Assay</td>
		</tr>
		<tr>
			<td>EDTA-Na2</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>Mobile Phase</td>
		</tr>
		<tr>
			<td>GraphPad Software</td>
			<td>Graphpad Software, Inc</td>
			<td></td>
			<td>Statistical Analysis</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>HPLC, Decade Amperometric</td>
			<td>Anetc</td>
			<td></td>
			<td>HPLC, LC-EC system</td>
		</tr>
		<tr>
			<td>HPLC</td>
			<td>Amuza</td>
			<td></td>
			<td>HPLC HTEC-510.</td>
		</tr>
		<tr>
			<td>L-Asrobic Acid</td>
			<td>Sigma Aldrich</td>
			<td>A5960</td>
			<td>Dissection Buffer</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma</td>
			<td>7487-88-9</td>
			<td>KH Buffer</td>
		</tr>
		<tr>
			<td>Microcentrifuge Filter Units UltraFree</td>
			<td>Millipore</td>
			<td>C7554</td>
			<td>Assay - 6 to fit in 48 well plate</td>
		</tr>
		<tr>
			<td>MTT</td>
			<td>Thermo Fisher</td>
			<td>M6494</td>
			<td>MTT Assay</td>
		</tr>
		<tr>
			<td>Nanosep</td>
			<td>VWR</td>
			<td>29300-606</td>
			<td>Assay; protein assay</td>
		</tr>
		<tr>
			<td>Octanesulfonic acid</td>
			<td>Sigma Aldrich</td>
			<td>V800010</td>
			<td>Mobile Phase</td>
		</tr>
		<tr>
			<td>Pargyline Clorohydrate</td>
			<td>Sigma Aldrich</td>
			<td>P8013</td>
			<td>Modified Artifical Cerebrospinal Fluid OR Efflux Buffer</td>
		</tr>
		<tr>
			<td>Phosphoric Acid</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>Mobile Phase</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>12636</td>
			<td>KH Buffer</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Sigma</td>
			<td>1001655559</td>
			<td>KH Buffer</td>
		</tr>
		<tr>
			<td>Precisonary VF-21-0Z</td>
			<td>Precissonary</td>
			<td></td>
			<td>Compresstome</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma Aldrich</td>
			<td>P2714</td>
			<td>Lysis buffer.</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>Dissection Buffer</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td>Dissection Buffer</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td>KH Buffer</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>L3771</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787</td>
			<td>MTT Assay / Lysis buffer</td>
		</tr>
	</tbody>
</table>

a plate-based assay for the measurement of endogenous monoamine release in acute brain slices

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo&#160;microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

This technique describes the use of brain slices to measure the release of endogenous monoamines using HPLC with electrochemical detection based in a 48-well plate with an internal tissue holder. Experimental set up is depicted in Figure 1 and Figure 2. Initially, to ensure tissue viability by the end of the experimentation, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay was performed. Af...

Watch this Scientific Journal Video about A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices at JoVE.com

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

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in ...

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.

Research

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