Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

Summary

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

Abstract

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

Introduction

Brain dopamine (DA) has been implicated in central responses to intake of palatable sugars through proposed hedonic^1,2, effort-related³ and habit-based^4,5 mechanisms of action. The primary DA pathway implicated in these effects originates in the ventral tegmental area (VTA), and projects to the nucleus accumbens (NAC) core and shell, the basolateral and central-cortico-medial amygdala (AMY), and the prelimbic and infralimbic medial prefrontal cortex (mPFC) (see reviews^6,7). The VTA has been implicated in sucrose intake^8,9, and DA release is observed following sugar intake in the NAC^10-15, AMY

Protocol

These experimental protocols have been approved by the Institutional Animal Care and Use Committee certifying that all subjects and procedures are in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.

1. Subjects

1. Purchase and/or breed male Sprague-Dawley rats (260 - 300 g).
2. House rats individually in wire mesh cages. Maintain them on a 12:12 hr light/dark cycle with rat chow and water available ad libitum.
3. Assign appropriate sample sizes (e.g., n ≈ 6 - 8) randomly into groups.

2. Testing Apparatus and Intake Procedures

1. Us....

Results

All representative results described below have been published previously⁶⁹, and are re-presented here to support "proof of concept" in indicating the effectiveness of the technique.

Solution Intakes
Significant differences in baseline saccharin intakes were observed over the first four days for all animals (F(3,108) = 57.27, p < 0.001) with intakes (Day 1: 1.3 (± 0.2) ml; Day 2: 3.9 (± 0.4) ml.......

Discussion

The goal of the study was to determine if the source (VTA) and forebrain projection targets (NAC, AMY, mPFC) of DA reward-related neurons were simultaneously activated after novel ingestion of fat and sugar in rats using the cellular c-fos technique. The present study is a detailed description of the protocols of a study published previously⁶⁹. It was hypothesized that the VTA, its major projection zones to the prelimbic and infralimbic mPFC, the core and shell of the NAC and the basolateral and central-cortic.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
Sprague-Dawley rats	Charles River Laboratories	CD-1
Wire Mesh Cages	Lab Products, Seaford, DE	30-Cage rack
Rat Chow	PMI Nutrition International	5001
Taut Metal Spring	Lab Products, Seaford, DE	n/a
Rat Weighing Scale	Fisher Scientific Company	n/a
Nalgene Centrifuge Tubes	Lab Products, Seaford, DE	10-0501
Rubber Stopper	Lab Products, Seaford, DE	n/a
Metal Sippers	Lab Products, Seaford, DE	n/a
Saccharin	Sigma Chemical Co	82385-42-0
Kool-Aid, Cherry	Kool-Aid	Commerical
Kool-Aid, Grape	Kool-Aid	Commercial
Fructose	Sigma Chemical Co	F0127
Glucose	Sigma Chemical Co	G8270
Corn Oil	Mazzola	Commerical
Xanthan Gum	Sigma Chemical Co	11138-66-2
Sliding Microtome	Microm International	n/a
Neurolucida Camera	MBF Bioscience	Software application
Gelatin-coated Slides	Fisher Scientific Company	12-550-343
Cover glass	Fisher Scientific Company	12-545-M
Golden Nylon Brushes	Loew-Cornell	2037
Natural Hair Sable	Loew-Cornell	2022
24 Well Plates	Fisher Scientific	3527
6 Well Plates	Fisher Scientific	3506
1 L Pyrex bottles	Fisher Scientific	1395-1L
Tissue insert (tissue strainer)	Fisher Scientific	7200214
Eagle pipettes	World Precision Instruments	E10 for 1-10μl
Eagle pipettes	World Precision Instruments	E100 for 20-100μl
Eagle pipettes	World Precision Instruments	E200 for 50-200μl
Eagle pipettes	World Precision Instruments	E1000 for 100-1000μl
Eagle pipettes	World Precision Instruments	E5000 for 1000-5000μl
Universal Tips .1-10 μl	World Precision Instruments	500192
Universal Tips 5-200 μl	World Precision Instruments	500194
Universal Tips 500-5,000 μl	World Precision Instruments	500198
Blade Vibroslice 100	World Precision Instruments	BLADE
DPX Mounting Medium	Electron Microscopy	13510
15 ml centrifuge tubes	Biologix Research Co.	10-0501
Slide Boxes	Biologix Research Co.	41-6100
Orbital Shaker	Madell Corporation	ZD-9556
weigh boats	Fisher Scientific	02-202-100
5 ml disposable pipettes	Fisher Scientific	13-711-5AM
Stereo Investigator Software	Micro Bright Field	Software application
Name	Company	Catalog number	Comments
Reagents
Paraformaldehyde Granular	Fisher Scientific	19210
NaCl	Fisher Scientific	S271-1
Sodium Phophate Monobasic	Fisher Scientific	S468-500
Sodium Phosphate Diphasic	Fisher Scientific	BP332-500
Hydrogen Peroxide	Fisher Scientific	H324-500
SafeClear II	Fisher Scientific	23-044-192
Methanol	Fisher Scientific	A412-1
Normal Goat Serum	Vector	S-1000
Biotinylated Anti-Rabbit IgG (H+L)	Vector	BA-1000
ABC Kit Peroxidase Standard	Vector	PK-4000
Anti-cFos (Ab-5) Rabbit	EMD chem/Cal Biochem	PC38
Triton X 100	SigmaAldrich	X-100
3,3' diaminobenzidine tetra hydrochloride	SigmaAldrich	D5905
Sodium Hydroxide	SigmaAldrich	5881
Primary TH anti body	EMD Millipore	AB152
Euthosol	Virbac AH