Onderzoek van Rapid Dopamine Dynamics met Fast scan cyclische voltammetrie Tijdens intra-orale Tastant Administration in Awake Rats

Summary

Rapid fluctuations in extracellular dopamine (DA) mediate both reward processing and motivated behavior in mammals. This manuscript describes the combined use of fast scan cyclic voltammetry (FSCV) and intra-oral tastant administration to determine how tastants alter rapid dopamine release in awake, freely moving rats.

Abstract

Rapid, phasic dopamine (DA) release in the mammalian brain plays a critical role in reward processing, reinforcement learning, and motivational control. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique with high spatial and temporal (sub-second) resolution that has been utilized to examine phasic DA release in several types of preparations. In vitro experiments in single-cells and brain slices and in vivo experiments in anesthetized rodents have been used to identify mechanisms that mediate dopamine release and uptake under normal conditions and in disease models. Over the last 20 years, in vivo FSCV experiments in awake, freely moving rodents have also provided insight of dopaminergic mechanisms in reward processing and reward learning. One major advantage of the awake, freely moving preparation is the ability to examine rapid DA fluctuations that are time-locked to specific behavioral events or to reward or cue presentation. However, one limitation of combined behavior and voltammetry experiments is the difficulty of dissociating DA effects that are specific to primary rewarding or aversive stimuli from co-occurring DA fluctuations that mediate reward-directed or other motor behaviors. Here, we describe a combined method using in vivo FSCV and intra-oral infusion in an awake rat to directly investigate DA responses to oral tastants. In these experiments, oral tastants are infused directly to the palate of the rat – bypassing reward-directed behavior and voluntary drinking behavior – allowing for direct examination of DA responses to tastant stimuli.

Introduction

Phasic DA release plays an important role in mediating reward-directed behavior ^[1-3]. However, isolating and studying how a primary reward alters phasic DA release is often complicated by co-occurring behavioral or cognitive processes that are also capable of altering phasic DA release - such as decision making processes or reward-directed motor behavior to acquire the reward. In the current work, we isolate phasic DA responses to tastants through the use of in vivo fast-scan cyclic voltammetry (FSCV) combined with tastant delivery through intraoral cannulae. This technique bypasses choice and action and allows us to examine extracellular DA relea....

Protocol

Alle experimenten werden uitgevoerd volgens de National Institutes of Health (NIH) Gids voor de Zorg en gebruik van proefdieren en werden goedgekeurd door de Yale University Institutional Animal Care en gebruik Comite (IACUC).

1) Pre-operatieve Voorbereidingen

1. Orale oplossing voorbereiding
   1. Maak oplossingen van 10% sucrose en 0,005% L-menthol in gedeïoniseerd water (pH 7,4). Bewaar deze oplossingen in gesloten containers, bij KT, en remake elke 2 weken, om degradatie te voorkomen.
2. Elektrode ijkoplossingen
   1. Maak Tris buffer (pH 7,4) op elk moment voorafgaand aan de kalibratie. De oplossing bes....

Results

FSCV gecombineerd met intra-orale katheter implantatie werd gebruikt om te onderzoeken hoe sucrose, een appetitive tastant, moduleert fasische DA release in de NAc kern. Vóór tastant infusie elektrische stimulatie (150 uA, 60 Hz, 24 pulsen, aangeduid met het rode balk) van het VTA produceert sterke toename driefase DA afgifte in de NAc (figuur 1) Figuur 1 toont een kleurenplot met potentiaal op. de y-as de tijd op de x-as en stroom (weergegeven als valse kleur) op de z-as. Onder de kleur plot is DA conc.......

Discussion

Intraorale tastant levering gecombineerd met FSCV maakt analyse van de "real-time" DA reacties op orale smaakstoffen. Er zijn drie belangrijke stappen in het protocol die zijn vereist voor succesvolle DA metingen. Ten eerste goede implantatie van de orale katheter kritisch voor afgifte van smaakstoffen. Ervoor zorgen dat de katheter achter de eerste kies is geplaatst en ingeklemd in plaats voorkomt dat de catheter verliest doorgankelijkheid en voorkomt onbedoelde verwijdering door het dier. Spoelen van de kath.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stimulating electrode	PlasticsOne	MS303/2-A/SPC	when ordering, request a 22 mm cut below pedestal
Cannula for electrode	BioAnalytical Systems	MD-2251
Glass capillary	A-M systems	624503
Carbon fiber	Thornel	T650
Electrode puller	Narishige International	PE-22
Neurolog stimulus isolator	Digitimer Ltd.	DS4
Heat shrink	3M	FP-301
Insulated wires for electrodes	Squires electronics	Custom	L 3.000 x 1.000s x 1.000s UL1423 30/1 BLU
Micromanipulator	Univ. of Illinois at Chicago, Engineering Machine Shop
Epoxy	ITW Devcon	14250	"5 minute epoxy"
Copolymer of perfluoro-3,6-dioxa-4-methyl-7octene-sulfonic acid and tetrafluoroethylene	Ion Power	LQ-1105	i.e., Nafion
Silver paint	GC Electronics	22-023
Power supply	BK Precision	9110
Tubing for intra-oral catheters	Intramedic	427426	PE 100, I.D. = 0.86 mm; O.D. = 1.52 mm
Tubing for intra-oral infusion	Fischer Scientific	02-587-1A	I.D. 1/32"; O.D. 3/32"
Syringe pump for flow cell	Pump Systems Inc.	NE 1000
Surgical cement	Dentsply Caulk	675571 and 675572
Air actuator	VICI	A60	6 position digital valve interface
Digital valve interface	VICI	DVI	230 VAC
Quad headstage	Univ. of N. Carolina, Electronics Facility
UEI power supply	Univ. of N. Carolina, Electronics Facility
UEI breakout box	Univ. of N. Carolina, Electronics Facility
Power supply for tastant syringe pump	Med Associates	SG-504
Tastant syringe pump	Med Associates	PHM-107
Tungsten microelectrode	MicroProbes	WE30030.5A3
Silver wire reference with AgCl	InVivo Metric	E255A
Sucrose	Sigma	80497
Magnesium chloride	Sigma	M8266
Sodium chlroide	Sigma	S7653
Perchloric acid	Sigma	244252
Hydrochloric acid (4 M)	Sigma	54435
Soidum hydroxide	Sigma	306576
Hydrogen peroxide	Sigma	H1009
Dopamine hydrochloride	Sigma	H8502
TarHeel HDCV Software	University of North Carolina-Chapel Hill		Must request software: click here for link to software request page