Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Summary

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

Abstract

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 μm) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Protocol

1. Experimental essentials

Electrode Fabrication

• There are numerous carbon fiber microelectrodes fabrication methods since most are made in-house. Typically what dictates the electrode fabrication details is the electrochemical technique that is applied to the electrode (e.g. amperometry vs. FSCV). For FSCV, microelectrodes can be made in-house using the following three-step procedure. For a more complete description of carbon fiber electrode fabrication, see a recent JOVE article ¹. However, note that the electrodes described below are cylindrical carbon fiber microelectrodes, which require fewer steps to ....

Discussion

The protocol presented here demonstrates how to prepare and use mouse coronal brain slices for FSCV experiments. Although this method is specific to obtaining and measuring dopamine dynamics, other neurotransmitters such as adenosine, hydrogen peroxide, norepinephrine, and serotonin have been monitored in vivo or in vitro with FSCV ^{3, 8 - 11}. FSCV can be used to monitor some of these other neurochemicals by simple modifications of the waveform applied to the working electrode ^{3, 11}.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Potassium Chloride	Fisher Scientific	7447407
Sodium chloride	EMD Millipore	7647145
Magnesium chloride	Fisher Scientific	7791186
Calcium chloride	Fisher Scientific	10035048
Sodium bicarbonate	EMD Millipore	144558
Sodium phosphate,Dibasic	EMD Millipore	7558794
D-glucose	Fisher Scientific	50997
Ascorbic acid	Fisher Scientific	50817
Sucrose	Fisher Scientific	57501
Carbon fiber	Goodfellow
Glass capillary	A-M Systems	602000
Silver wire	A-M Systems	787000
Tungsten stimulating electrode	Plastics One
Platinum wire
Lead wire	Squires Electronics, Cornelius, OR
Loctite 404 instant adhesive	Hankal Corp.
Razor blade	World Precision Instruments, Inc.
BD Spinal needle	BD Biosciences	REF 405234
Surgical Blade	Feather Safety Razor Co, Ltd.
TH software	ESA Inc.,Chelmsford, MA
Submersion recording chamber	Custom Scientific
Neorolog stimulus isolator	Digitimer Ltd.	NL800A
Automatic temperature controller	Warner Instruments
Microscope (SZX7)	Olympus Corporation
Microscope	Fisher Scientific
Vibratome 3000 sectioning system	St. Louis , MO.
Perfusion pump	Watson-Marlow Pumps Group	H110708
Micropipette puller	Narishige International
ChemClamp potentiostat	Dagan Corporations