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Экспертиза Rapid допамина Dynamics с быстрого сканирования циклической вольтамперометрии Во интраоральных Tastant администрации в Пробудитесь крыс

Published: August 12, 2015
doi:

Özet

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 release during direct infusion of a tastant to the palate of a rat.

FSCV is an electrochemical technique with high temporal and spatial resolution, permitting measurements of DA release in a discrete local area (approximately 100 µm) on a sub-second scale (10 Hz resolution). The combination of intra-oral delivery and FSCV provides the advantage of observing rapid, ‘real-time’ DA responses to a tastant, which cannot be examined using conventional microdialysis methods. Furthermore, phasic DA release in response to either rewards or reward-associated cues occurs at concentrations between 20-100 nM, which is above the 10-20 nM detection threshold for FSCV [4]. The high spatial resolution of FSCV also permits recording from sub-regions of small brain areas, such as the nucleus accumbens core (NAc). Thus, FSCV combined with intraoral infusions of tastants is an ideal model for studying how tastants or other stimuli alter phasic DA release in an awake, behaving animals. Indeed, these techniques have permitted experiments investigating how rewarding and aversive tastants alter extracellular DA release [5].

Over the last decade, FSCV analyses have been successfully combined with intravenous drug delivery [6] and rat self-administration paradigms [7, 8] to identify the role of phasic DA mechanisms in drug addiction models. In addition, combined intraoral delivery with FSCV has been used to examine how tastant cues paired with cocaine availability modulate phasic DA release and behavioral responses that reflect emotional affect [3]. This combined intraoral and FSCV methodology can also be powerfully utilized to examine phasic DA responses to flavorants, such as menthol and oral sweeteners, that are added to cigarettes and dissolvable tobacco products [9-11]. Although many of the flavorants added to tobacco products are appetitive [9-11], it is unknown if these flavorants increase phasic DA release in a manner consistent with a rewarding hedonic valence. Indeed, flavorants added to cigarettes and to dissolvable tobacco products may have direct effects on the DA reward system and may act through dopaminergic mechanisms to influence the rewarding valence of cigarettes and other tobacco products. Thus, intraoral delivery combined with FSCV can provide new understanding on how flavorants modulate rapid DA release. The use of combined intra-oral and in vivo FSCV methodology, and the data obtained from such studies, can also facilitate future studies to determine how flavorants and nicotine interact to alter DA signaling and to potentially modulate nicotine reinforcement. Further, the data gained from such studies can be used to inform regulatory decisions about tobacco product flavorants.

Protocol

Все эксперименты были проведены в соответствии с Национальными Институтами (NIH) Руководство здравоохранения для ухода и использования лабораторных животных и были одобрены Институциональные уходу и использованию животных комитета Йельского университета (IACUC) по. 1) Пре?…

Representative Results

FSCV в сочетании с внутриротовой катетера имплантации был использован для изучения, как сахароза, в аппетитивную tastant, модулирует фазовое DA-релиз в ядре НАК. До tastant инфузии, электрической стимуляции (150 мкА, 60 Гц, 24 импульсов, с указанием красной полосой) в VTA производит надежные увеличение …

Discussion

Интраоральная доставка tastant в сочетании с FSCV позволяет анализ "реального времени" ответов на устные DA ароматизаторами. Есть три важных шагов в протоколе, которые необходимы для успешных измерений DA. Во-первых, собственно имплантация полости катетера имеет решающее значение для до…

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

Research reported in this publication was supported by an NSF Graduate Research Fellowship (RJW) and by the National Institute on Drug Abuse of the National Institutes of Health and FDA Center for Tobacco Products (CTP) under Award Number P50DA036151(EJN and NAA). The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.

Materials

Name of Material/ Equipment Company Catalog Number Comments/Description
Stimulating electrode PlasticsOne MS303/2-A/SPC (Stimulating electrode) when ordering, request a 22mm cut below pedastal 
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 (Wire for electrodes) L 3.000 x 1.000s x 1.000s UL1423 30/1 BLU    
Micromanipulator Univ. of Illinois at Chicago, Engineering Machine Shop n/a
Epoxy ITW Devcon 14250 (Epoxy) "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 (Tubing for intra-oral catheters) PE 100, I.D.=0.86mm; O.D.=1.52mm
Tubing for intra-oral infusion Fischer Scientific 02-587-1A (Tubing for intra-oral infusion) 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 acuatator VICI A60 (Air actuator) 6 position digital valve interface
Digital Valve Interface VICI DVI (Digital Valve Interface) 230 VAC
Quad Headstage Univ. of N. Carolina, Electronics Facility n/a
UEI Power Supply Univ. of N. Carolina, Electronics Facility n/a
UEI Breakout Box Univ. of N. Carolina, Electronics Facility n/a
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 (4M) 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

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Bu Makaleden Alıntı Yapın
Wickham, R. J., Park, J., Nunes, E. J., Addy, N. A. Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats. J. Vis. Exp. (102), e52468, doi:10.3791/52468 (2015).

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