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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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-firing2,3,4,5. 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.......

Representative Results

CIS-FSCV was used to study the function of VTA N-methyl-D-aspartate receptors (NMDAR), nicotinic acetylcholine receptors (nAChRs), and muscarinic acetylcholine receptors (mAChRs) in driving phasic DA release in the NAc core. Figure 2 shows representative data for a negative control, infusion of 0.9% saline, before (baseline) and 9 min postinfusion (saline). Figure 2 shows a color plot with potential on the y-axis, time on the x-a.......

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, .......

Acknowledgements

Work was supported by Elizabethtown College (R.J.W, M.L., and L.M.), by a NSF Graduate Fellowship (R.J.W.) and by the Yale School of Medicine (N.A.).

....

Materials

NameCompanyCatalog NumberComments
Electrode Filling Solution/Supplies
MicropipetteWorld Precision InstrumentsMF286-5 (28 gauge)
Potassium AcetateSigma236497-100G
Potassium ChlorideSigmaP3911-25G
Electrode Supplies
Carbon fiberThornelT650
Electrode pullerNarishige InternationalPE-22Note: horizontal pullers can be used as well
Glass capillaryA-M systems626000
Insulated wires for electrodesWeico Wire and Cable IncorporatedUL 1423Length; 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 ScientificM3700
PinPhoenix EnterprisesHWS1646To be soldered onto the insuled electrode wire and reference electrode; connects to headstage
PuttyAlcolin23922-1003Used to place electrode on while cutting the carbon fiber
Scalpal BladeWorld Precision Instruments500239For cutting carbon fiber to the apprpriate length
Silver WireSigma327026-4G
FSCV Hardware/Software
Faraday CageU-LineH-3618 (36" x 24" x 42")
PotentiostatUniv. of N. Carolina, Electronics Facility
Stimulating electrodePlasticsOneMS303/2-A/SPCwhen ordering, request a 22 mm cut below pedestal
TarHeel HDCV SoftwareUniversity of North Carolina-Chapel Hill-https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/ for ordering information
UEI breakout boxUniv. of N. Carolina, Electronics Facilityhttps://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/ for ordering information
UEI power supplyUniv. of N. Carolina, Electronics Facilityhttps://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/ for ordering information
Stimulator Hardware
Neurolog stimulus isolatorDigitimer Ltd.DS4Neurolog 800A
Infusion/Stimulation Supplies
Infusion PumpNew Era Syringe PumpNE-300
Internal CannulaPlasticsOneC315I/SPC INTERNAL 33GA
Microliter SyringeHamilton80308
TubingPlasticsOneC313CT/ PKG TUBING 023 X 050 PE50
Surgical Supplies
Cannula HolderKopf Instruments1776 P-1
Cotton Tip ApplicatorsVitality Medical806
Electrode HolderKopf Instruments1770
Heating PadKent ScientificRT-0501
Povidone IodineVitality Medical29906-004
ScrewsStoeltingBone Anchor Screws/Pkg.of 1001.59 mm O.D., 3.2 mm long
Silver wire reference with AgClInVivo MetricE255A
Square GauzeVitality Medical441408
StereotaxKopf InstrumentsModel 902 (Dual Arm Bar)
Histological Supplies
FormulinSigma1004960700
Power supplyBK Precision9110
SucroseSigma80497
Tungsten microelectrodeMicroProbesWE30030.5A3
Drugs for infusions
((2R)-amino-5-phosphonovaleric acidSigma AldrichA5282
N-methyl-D-aspartateSigma AldrichM3262
Mecamylamine hydrochloride (M9020-5mg)Sigma AldrichM9020
Scopolamine hydrobromide (S0929-1g)Sigma AldrichS0929

References

  1. Grace, A. A., Bunney, B. S. The control of firing pattern in nigral dopamine neurons: burst firing. Journal of Neuroscience. 4 (11), 2877-2890 (1984).
  2. Lester, D. B., et al. Midbrain acetylcholine and ....

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