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^16,17 and mPFC^18-20. Fat intake also stimulates DA NAC release²¹, and another DA-rich projection zone to the dorsal striatum (caudate-putamen) has been also associated with DA-mediated feeding^22,23. Kelley^24-27 proposed that these multiple projection zones of this DA-mediated system formed an integrated and interactive distributed brain network through extensive and intimate interconnections^28-34.

In addition to the ability of DA D1 and D2 receptor antagonists to reduce intake of sugars^35-37 and fats^38-40, DA signaling has also been implicated in mediating the ability of sugars and fats to produce conditioned flavor preferences (CFP)^41-46. Microinjections of a DA D1 receptor antagonist into the NAC, AMY or mPFC^47-49 eliminate acquisition of CFP elicited by intragastric glucose. Whereas microinjections of either DA D1 or D2 receptor antagonists into the mPFC eliminates acquisition of fructose-CFP⁵⁰, the acquisition and expression of fructose-CFP are differentially blocked by DA antagonists in the NAC and AMY^51,52.

The c-fos technique^53,54 has been employed to investigate neural activation induced by palatable intake and neural activation. The term "c-fos activation" will be used throughout the manuscript, and is operationally defined by increased transcription of c-Fos during neuronal depolarization. Sucrose intake increased fos-like immunoreactivity (FLI) in the central AMY nucleus, the VTA as well as the shell, but not core, of the NAC^55-57. Whereas sucrose intake in sham-feeding rats significantly increased FLI in the AMY and the NAC, but not the VTA⁵⁸, intragastric sucrose or glucose infusions significantly increased FLI in the NAC and central and basolateral nuclei of the AMY^59,60. Repeated addition of sucrose to scheduled chow access increased FLI in the mPFC as well as the NAC shell and core⁶¹. A sucrose concentration downshift paradigm revealed that the greatest FLI increases occurred in the basolateral AMY and NAC, but not the VTA⁶². Following conditioning, extinction of sugar-related natural reward behaviors increased FLI in the basolateral AMY and the NAC⁶³. Moreover, pairing sugar availability to a tone resulted in the tone subsequently increasing FLI levels in the basolateral AMY⁶⁴. High-fat intake also increased FLI in NAC and mPFC sites^65-67.

Most of the previously cited studies examined sugar and fat effects on c-fos activation in single sites that do not provide information about identification of reward-related distributed brain networks^24-27. Further, many of the studies also did not delineate the relative contributions of sub-areas of the NAC (core and shell), AMY (basolateral and central-cortico-medial) and mPFC (prelimbic and infralimbic) that could potentially be examined by the advantage of excellent spatial, single-cell resolution in c-Fos mapping⁶⁸. Our laboratory⁶⁹ recently used c-fos activation and simultaneously measured alterations in the VTA DA pathway and its projection zones (NAC, AMY and mPFC) after novel ingestion of fats and sugars in rats. The present study describes the procedural and methodological steps to simultaneously analyze whether acute exposure to six different solutions (corn oil, glucose, fructose, saccharin, water and a fat emulsion control) would differentially activate FLI in sub-areas of the NAC, AMY, mPFC as well as the dorsal striatum. This simultaneous detection of differences allowed confirmation of significant effects on FLI in each site and determination as to whether changes in one particular site correlated with changes in related sites, thereby providing support for a distributed brain network^24-27. These procedures tested whether the VTA, the prelimbic and infralimbic mPFC, the core and shell of the NAC, and the basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of glucose (8%), fructose (8%), corn oil (3.5%) and saccharin (0.2%) solutions.

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. Use calibrated centrifuge tubes with rubber stoppers and a 45° angle metal sipper tube to provide accurate measurement (± 0.1 ml) of the presented solutions. Secure them to the home cages by a taut metal spring to allow visibility of the calibrations.
2. Restrict food rations (~ 15/g/day) of the rats to reduce weight to 85% of their original body weight to increase motivation to consume the solutions. Note: The weight reduction should take between 3 - 5 days.
3. Provide pre-training solutions (10 ml) of 0.2% saccharin for four days over a 1 hr session to maximize the probability that rats will sample the subsequent test solutions with short (less than 1 min) latency.
4. Confirm flow through the centrifuge tube by spilling a few drops.
5. Weigh tubes before and after each session to obtain intake measurement.
6. Perform an intake test on the fifth day on subgroups receiving one of six solutions (10 ml, 1 hr): a) water, b) novel flavored (0.05% cherry flavor) 0.2% saccharin, c) 8% fructose, d) 8% glucose, e) 3.5% corn oil suspended in 0.3% xanthan gum, and f) 0.3% xanthan gum.
7. Ensure that the nutrient solutions are isocaloric; thus, the 3.5% corn-oil concentration is isocaloric to the 8% sugar solutions.
8. Ensure that rats sample solutions with short latency (less than 1 min). If this requirement is not met, then discard the subject from the study.

3. Tissue Preparation

1. Anesthetize each animal by an intraperitoneal injection of pentobarbital 90 min after initial exposure to each test solution. Confirm that animals are properly anesthetized by demonstrating that the animal is no longer responsive to such reflexes as withdrawal to foot pinch, blinking following direct corneal pressure or head shaking to deep pinna stimulation.
2. Perfuse each animal transcardially as described previously⁶⁹.
   1. Anesthetize rats with an overdose of sodium pentobarbital (65 mg/kg), remove the ribcage and expose the chest for free access to the heart⁶⁹.
   2. Place the needle in the apex of the left heart valve, and cut the vena cava. Administer phosphate buffer solution (PBS, ~ 180 ml) followed by a phosphate-buffered fixative containing 4% paraformaldehyde (~ 180 ml).
   3. Ensure that the animal actually is being perfused correctly by examining whether liquid is leaving other cavities, such as the nose, mouth, and genital areas. Note: Proper fixation with paraformaldehyde will be accompanied by large muscle movements. If this does not occur, re-adjust the needle until this reaction occurs.
3. Remove the brain from the skull quickly by cutting fur and skin away from the skull. Use rongeurs to crack and remove the bone from the brain moving from rear to front. Work initially in the area below and behind the cerebellum, ensuring that the rongeur is between the bone and meningeal pia mater. Once the top and sides of the skull are removed, use a small spatula to lift the brain from the base, and snip cranial nerves with small scissors. Be careful not to damage the brain while attempting to remove the bone.
4. Fix the brains in a 4% paraformaldehyde solution overnight at 4 °C. Place the brains in a 30% sucrose/70% PBS solution at room temperature until they settle at the bottom of the container.
5. Block the brain
   1. Remove the rostral part of the brain cutting transversely caudal to the olfactory bulb.
   2. Remove the caudal part of the brain cutting transversely at the level of the cerebellum and the pons.
6. Mount the brain coronally with the caudal part fixed to the stage of a sliding microtome, and cut coronal sections (40 µm) through the mPFC (+2.86 – +2.20 mm rostral to bregma), the NAC core and shell and dorsal striatum (+1.76 – +1.60 mm rostral to bregma), the AMY (-2.12 – -2.92 mm caudal to bregma), and the VTA (-5.20 – -5.60 mm caudal to bregma). Use a rat brain atlas⁷⁰ for guidance.
7. Collect free-floating sections into individual wells of a 24-well plate filled with PBS for eventual immunohistochemical analysis⁷¹. Use Parafilm to seal the 24 well plate to make sure the PBS does not evaporate in the container and dry up the brain. Store the brain tissue in 4 °C.

4. c-fos Procedures (Adapted from ⁷¹)

1. Treat each section with 5 ml of 5% Normal Goat Serum and 0.2% Triton x-100 in PBS for 1 hr.
2. Incubate the treated sections with primary antibodies (rabbit anti-c-fos, 1:5,000) at 4 °C for 36 hr in wells containing 1 ml of PBS.
3. Rinse sections 3x with PBS (5 ml) for 10 min each.
4. Incubate with secondary antibodies (biotinylated goat anti-rabbit; 1:200) at RT for 2 hr in wells containing 1 ml of PBS.
5. Rinse each section 3x in PBS (5 ml) for 10 min each.
6. Incubate the rinsed sections for 2 hr in a commercially available avidin-horseradish peroxidase mixture that comes in a kit composed of Avadin DH (100 µl) and Biotinylated Horseradish Peroxidase H (100 µl) in 5 ml of PBS.
7. Re-rinse the sections 3x in PBS (5 ml) for 10 min each.
8. React the sections with 0.05% diaminobenzidine (DAB) in the presence of 0.0015% H₂O₂ for 5 - 10 min, depending on the reactivity of the tissue in wells containing 5 ml of the DAB solution.
9. Double-label the VTA sections. Incubate them with a tyrosine hydroxylase (TH) antibody (rabbit anti-rat TH, 1:2,000) in PBS (5 ml) overnight at 4 °C.
10. Rinse sections 3x in PBS (5 ml) for 10 min each.
11. Incubate with secondary antibodies (biotinylated goat anti-rabbit; 1:200) in PBS (5 ml) at room temperature for 2 hr.
12. Rinse sections 3x in PBS (5 ml) for 10 min each.
13. Visualize the antibodies by using a secondary antibody-peroxidase complex. React with a combination of 0.05% DAB and a 0.3% nickel sulfate solution, for 5 - 10 min, depending on the reaction of the tissue in wells containing 5 ml of the DAB/NiCl solution.
14. Ensure that the DAB solution is milky light green in color from its reaction with the 0.3% nickel sulfate. If the solution is too green, then the reaction will be too dark.
15. Mount all sections onto gelatin-coated slides. Let them dry overnight, and then cover-slip with a few drops of a Toluene-Based Solution (TBS).
16. Code slides so that the experimental condition is unknown to the observers.

5. Determination of c-fos Immunoreactive Counts

1. Assign pairs of unbiased observers to count Fos-positive neurons in these regions of interest (ROI): prelimbic mPFC, infralimbic mPFC, NAC core, NAC shell, basolateral AMY nuclei, central-cortico-medial AMY, dorsal striatum, and VTA. Delineate whether c-Fos immunoreactivity was present in TH+ and TH- cells in the VTA. Figure 1 provides a screen-captured picture of the NAC from the microscope.

Figure 1. Representative Section of the Nucleus Accumbens (NAC) Displaying Regions of Interest Outlined by the Grid by which c-fos Counts are Made. The NAC shell is found medially and ventrally to the NAC core. The NAC core encircles the anterior commissure (Anterior Comm.). The ventral extent of the lateral ventricle (Lateral Vent.) is visible. Please click here to view a larger version of this figure.

2. Analyze at least three representative slices per site common to all animals in all of the testing conditions.
3. Use software and an optical microscope to analyze the entire region for each ROI by tracing an outline (Figure 1).
   1. For a given site, open the application and click on the acquisition drop-down menu and click "Live Image". Bring the ROI into focus and click the screen to establish a reference point. Then trace the chosen brain region using the grid as a guide. Once the trace is completed, count cells (steps 5.3.1.1 - 5.3.1.3).
      1. Double-click the software icon. Go to the menu bar, click on "Acquisition" and then "Live Image". Bring the ROI into focus and click the screen to establish a reference point.
      2. Go to the grid toolbar and click "Display Grid" and "Use grid labels". Outline the ROI with a predetermined trace.
      3. Count all cells in each ROI area, select a "+" in the left hand sidebar to keep count of c-fos cells. Click each cell singly to register counts. Consider a cell positive for c-fos when a defined dark red circle is observed (Figure 1).
   2. Repeat this process for each site.
   3. Record counts in a lab notebook and on the computer for future analysis. Go to the menu bar, click on "File", "Save Data File" to save the tracing and counts.
4. Ensure that inter-rater reliability (using correlation of counts) of the two uninformed raters for each section in each ROI always exceeds 0.8.

6. Statistics

1. Evaluate baseline saccharin intakes over the first four days using a repeated-measures 1-way analysis of variance (ANOVA) comparing the saccharin intakes of Days 1, 2, 3 and 4⁶⁹.
2. Compare saccharin intake (Day 4) with test intakes (Day 5) of the six groups using a randomized-block 2-way ANOVA⁶⁹.
3. Use Tukey comparisons (p < 0.05) to determine individual significant effects⁶⁹.
4. Determine inter-rater reliability, and then use a common observer's counts.
5. Average c-fos counts for the three representative slices for each site⁶⁹.
   1. Perform a 1-way ANOVA of c-fos activation induced by intake of the six solutions (3.5% corn oil, 8% glucose, 8% fructose, 0.2% flavored saccharin, xanthan gum control and water) for the perilimbic mPFC⁶⁹.
   2. Repeat parallel analyses of the six groups for infralimbic mPFC, NAC core, NAC shell, basolateral AMY, central-cortico-medial AMY, VTA and dorsal striatum. Use Tukey comparisons (p < 0.05) to reveal individual significant effects⁶⁹.
6. Compare corn oil intake with both water intake and intake of its suspension agent, xanthan gum. Compare fructose and glucose intakes with both water intake and intake of the non-nutritive sweetener, saccharin.
7. Establish whether significant relationships between solution intakes and c-fos activation in each of the sites were observed using Bonferroni r correlations (p < 0.05).
   1. Systematically compare c-fos counts in the perilimbic and infralimbic pre-frontal cortex for each animal in the 3.5% corn oil group.
   2. Repeat systematically parallel analyses of each pair of the six sites (VTA, dorsal striatum, infralimbic mPFC, perilimbic mPFC, NAC core, NAC shell, basolateral AMY, central-cortico-medial AMY) for the 3.5% corn oil.
   3. Repeat systematically parallel analyses of these six sites for the other five experimental intake conditions (8% glucose, 8% fructose, 0.2% flavored saccharin, xanthan gum control and water).
8. Take advantage of the fact that the same animals within a solution condition were evaluated across all sites by determining significant relationships between c-fos activation across solutions and within each solution using Bonferroni r correlations (p < 0.05).

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