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

Summary

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.

Abstract

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 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.

Introduction

A plethora of neurological and psychiatric diseases are associated with dysregulation or insufficient maintenance of monoamine neurotransmitter (dopamine [DA], serotonin [5-HT], norepinephrine [NE]) homeostasis^1,2,3. These conditions include, but are not limited to, depression^1,2, schizophrenia², anxiety², addiction⁴, menopause^5,6,7, pain

Protocol

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 set up is functional for different developmental points, female rats, and other species. If using a smaller animal, such as mice, the experimenter may ....

Results

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.......

Discussion

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 animals^{13,20,41,42,58,64,65,66,67}.......

Materials

Name	Company	Catalog Number	Comments
48 Well plate	NA	NA	Assay
Acetonitrile	Fischer Scientific	A998-1	Mobile Phase
Calcium Chloride Ahydrous	Sigma Aldrich	C1016	Modified Artifical Cerebrospinal Fluid OR Efflux Buffer
Clarity Software	Anetc
Citric Acid	Sigma Aldrich		Mobile Phase
D-(+)-Glucose	Sigma	1002608421	Dissection Buffer
DMF	Sigma Aldrich	D4551	MTT Assay
EDTA-Na2	Sigma Aldrich		Mobile Phase
GraphPad Software	Graphpad Software, Inc		Statistical Analysis
Glycerol	Sigma Aldrich	G5516	Lysis buffer
HEPES	Sigma Aldrich	H3375	Lysis buffer
HPLC, Decade Amperometric	Anetc		HPLC, LC-EC system
HPLC	Amuza		HPLC HTEC-510.
L-Asrobic 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
MTT	Thermo Fisher	M6494	MTT Assay
Nanosep	VWR	29300-606	Assay; protein assay
Octanesulfonic acid	Sigma Aldrich	V800010	Mobile Phase
Pargyline Clorohydrate	Sigma Aldrich	P8013	Modified Artifical Cerebrospinal Fluid OR Efflux Buffer
Phosphoric Acid	Sigma Aldrich		Mobile Phase
Potassium Chloride	Sigma	12636	KH Buffer
Potassium Phosphate Monobasic	Sigma	1001655559	KH Buffer
Precisonary VF-21-0Z	Precissonary		Compresstome
Protease Inhibitor Cocktail	Sigma Aldrich	P2714	Lysis buffer.
Sodium Bicarbonate	Sigma	S5761	Dissection Buffer
Sodium Bicarbonate	Sigma Aldrich	S5761	Dissection Buffer
Sodium Chloride	Sigma	S3014	KH Buffer
Sodium Dodecyl Sulfate	Sigma Aldrich	L3771	Lysis buffer
Triton X-100	Sigma Aldrich	T8787	MTT Assay / Lysis buffer