Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

Summary

The goal of this protocol is to directly manipulate ventral tegmental area receptors to study their contribution to subsecond dopamine release.

Abstract

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement learning. Understanding how the diverse neuronal inputs into the VTA control phasic DA release can provide a better picture of the circuitry that controls reward processing and reinforcement learning. Here, we describe a method that combines intra-VTA cannula infusions of pharmacological agonists and antagonists with stimulation-evoked phasic DA release (combined infusion and stimulation, or CIS) as measured by in vivo fast-scan cyclic voltammetry (FSCV). Using CIS-FSCV in anesthetized rats, a phasic DA response can be evoked by electrically stimulating the VTA with a bipolar electrode fitted with a cannula while recording in the nucleus accumbens core. Pharmacological agonists or antagonists can be infused directly at the stimulation site to investigate specific VTA receptors' roles in driving phasic DA release. A major benefit of CIS-FSCV is that VTA receptor function can be studied in vivo, building on in vitro studies.

Introduction

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) plays a vital role in reward-related behaviors. VTA DA neurons switch from a tonic-like firing (3-8 Hz) to a burst-like firing (>14 Hz)¹, which produces phasic DA release in the NAc. The VTA expresses a variety of somatodendritic receptors that are well-positioned to control the switch from tonic to burst-firing^2,3,4,5. Identifying which of these receptors, and their respective inputs, control phasic DA release will d....

Protocol

All experiments were conducted according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by both Elizabethtown College and Yale University Institutional Animal Care and Use Committee (IACUC). This protocol is specific to the anesthetized rat preparation of utilizing CIS-FSCV.

1. Presurgical preparations

1. Electrode solution preparation
   1. To make the electrode backfill solution, prepare a solution of 4 M potassium.......

Discussion

CIS-FSCV provides a unique opportunity to investigate VTA receptor mechanisms underlying phasic DA release. There are two critical steps in order to ensure a proper recording. First, a stable baseline recording must be achieved, with little drift in the evoked DA signal. An important way to increase the likelihood of establishing a stable recording is to ensure that the electrode has had plenty of time to cycle at both 60 Hz and 10 Hz (typically 15 min at 60 Hz, and 10 min at 10 Hz). As the carbon fiber is being cycled, .......

Materials

Name	Company	Catalog Number	Comments
Electrode Filling Solution/Supplies
Micropipette	World Precision Instruments	MF286-5 (28 gauge)
Potassium Acetate	Sigma	236497-100G
Potassium Chloride	Sigma	P3911-25G
Electrode Supplies
Carbon fiber	Thornel	T650
Electrode puller	Narishige International	PE-22	Note: horizontal pullers can be used as well
Glass capillary	A-M systems	626000
Insulated wires for electrodes	Weico Wire and Cable Incorporated	UL 1423	Length; 10 cm; diameter,0.4mm; must get custom made; insulated material should cover 5 cm of the wire
Light Microscope (for viewing and cutting electrode)	Fischer Scientific	M3700
Pin	Phoenix Enterprises	HWS1646	To be soldered onto the insuled electrode wire and reference electrode; connects to headstage
Putty	Alcolin	23922-1003	Used to place electrode on while cutting the carbon fiber
Scalpal Blade	World Precision Instruments	500239	For cutting carbon fiber to the apprpriate length
Silver Wire	Sigma	327026-4G
FSCV Hardware/Software
Faraday Cage	U-Line	H-3618 (36" x 24" x 42")
Potentiostat	Univ. of N. Carolina, Electronics Facility
Stimulating electrode	PlasticsOne	MS303/2-A/SPC	when ordering, request a 22 mm cut below pedestal
TarHeel HDCV Software	University of North Carolina-Chapel Hill	-	https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/ for ordering information
UEI breakout box	Univ. of N. Carolina, Electronics Facility		https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/ for ordering information
UEI power supply	Univ. of N. Carolina, Electronics Facility		https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/ for ordering information
Stimulator Hardware
Neurolog stimulus isolator	Digitimer Ltd.	DS4	Neurolog 800A
Infusion/Stimulation Supplies
Infusion Pump	New Era Syringe Pump	NE-300
Internal Cannula	PlasticsOne	C315I/SPC INTERNAL 33GA
Microliter Syringe	Hamilton	80308
Tubing	PlasticsOne	C313CT/ PKG TUBING 023 X 050 PE50
Surgical Supplies
Cannula Holder	Kopf Instruments	1776 P-1
Cotton Tip Applicators	Vitality Medical	806
Electrode Holder	Kopf Instruments	1770
Heating Pad	Kent Scientific	RT-0501
Povidone Iodine	Vitality Medical	29906-004
Screws	Stoelting	Bone Anchor Screws/Pkg.of 100	1.59 mm O.D., 3.2 mm long
Silver wire reference with AgCl	InVivo Metric	E255A
Square Gauze	Vitality Medical	441408
Stereotax	Kopf Instruments	Model 902 (Dual Arm Bar)
Histological Supplies
Formulin	Sigma	1004960700
Power supply	BK Precision	9110
Sucrose	Sigma	80497
Tungsten microelectrode	MicroProbes	WE30030.5A3
Drugs for infusions
((2R)-amino-5-phosphonovaleric acid	Sigma Aldrich	A5282
N-methyl-D-aspartate	Sigma Aldrich	M3262
Mecamylamine hydrochloride (M9020-5mg)	Sigma Aldrich	M9020
Scopolamine hydrobromide (S0929-1g)	Sigma Aldrich	S0929