Thanks to Diana Icaza-Culaki, Cristal Sampson and Theologia Karagiorgis for their hard work on this project.

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
<ol>
	<li>Purchase and/or breed male Sprague-Dawley rats (260 - 300 g).</li>
	<li>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.</li>
	<li>Assign appropriate sample sizes (e.g., n &#8776; 6 - 8) randomly into groups.</li>
</ol>
2. Testing Apparatus and Intake Procedures
<ol>
	<li>Use calibrated centrifuge tubes with rubber stoppers and a 45&#176; angle metal sipper tube to provide accurate measurement (&#177; 0.1 ml) of the presented solutions. Secure them to the home cages by a taut metal spring to allow visibility of the calibrations.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Confirm flow through the centrifuge tube by spilling a few drops.</li>
	<li>Weigh tubes before and after each session to obtain intake measurement.</li>
	<li>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.</li>
	<li>Ensure that the nutrient solutions are isocaloric; thus, the 3.5% corn-oil concentration is isocaloric to the 8% sugar solutions.</li>
	<li>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.</li>
</ol>
3. Tissue Preparation
<ol>
	<li>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.</li>
	<li>Perfuse each animal transcardially as described previously69.
	<ol>
		<li>Anesthetize rats with an overdose of sodium pentobarbital (65 mg/kg), remove the ribcage and expose the chest for free access to the heart69.</li>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Fix the brains in a 4% paraformaldehyde solution overnight at 4 &#176;C. Place the brains in a 30% sucrose/70% PBS solution at room temperature until they settle at the bottom of the container.</li>
	<li>Block the brain
	<ol>
		<li>Remove the rostral part of the brain cutting transversely caudal to the olfactory bulb.</li>
		<li>Remove the caudal part of the brain cutting transversely at the level of the cerebellum and the pons.</li>
	</ol>
	</li>
	<li>Mount the brain coronally with the caudal part fixed to the stage of a sliding microtome, and cut coronal sections (40 &#181;m) through the mPFC (+2.86 &#8211; +2.20 mm rostral to bregma), the NAC core and shell and dorsal striatum (+1.76 &#8211; +1.60 mm rostral to bregma), the AMY (-2.12 &#8211; -2.92 mm caudal to bregma), and the VTA (-5.20 &#8211; -5.60 mm caudal to bregma). Use a rat brain atlas70 for guidance.</li>
	<li>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 &#176;C.</li>
</ol>
4. c-fos Procedures (Adapted from 71)
<ol>
	<li>Treat each section with 5 ml of 5% Normal Goat Serum and 0.2% Triton x-100 in PBS for 1 hr.</li>
	<li>Incubate the treated sections with primary antibodies (rabbit anti-c-fos, 1:5,000) at 4 &#176;C for 36 hr in wells containing 1 ml of PBS.</li>
	<li>Rinse sections 3x with PBS (5 ml) for 10 min each.</li>
	<li>Incubate with secondary antibodies (biotinylated goat anti-rabbit; 1:200) at RT for 2 hr in wells containing 1 ml of PBS.</li>
	<li>Rinse each section 3x in PBS (5 ml) for 10 min each.</li>
	<li>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 &#181;l) and Biotinylated Horseradish Peroxidase H (100 &#181;l) in 5 ml of PBS.</li>
	<li>Re-rinse the sections 3x in PBS (5 ml) for 10 min each.</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>Rinse sections 3x in PBS (5 ml) for 10 min each.</li>
	<li>Incubate with secondary antibodies (biotinylated goat anti-rabbit; 1:200) in PBS (5 ml) at room temperature for 2 hr.</li>
	<li>Rinse sections 3x in PBS (5 ml) for 10 min each.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Code slides so that the experimental condition is unknown to the observers.</li>
</ol>
5. Determination of c-fos Immunoreactive Counts
<ol>
	<li>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.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53897/53897fig1.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/53897/53897fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Analyze at least three representative slices per site common to all animals in all of the testing conditions.</li>
	<li>Use software and an optical microscope to analyze the entire region for each ROI by tracing an outline (Figure 1).
	<ol>
		<li>For a given site, open the application and click on the acquisition drop-down menu and click &#34;Live Image&#34;. 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).
		<ol>
			<li>Double-click the software icon. Go to the menu bar, click on &#34;Acquisition&#34; and then &#34;Live Image&#34;. Bring the ROI into focus and click the screen to establish a reference point.</li>
			<li>Go to the grid toolbar and click &#34;Display Grid&#34; and &#34;Use grid labels&#34;. Outline the ROI with a predetermined trace.</li>
			<li>Count all cells in each ROI area, select a &#34;+&#34; 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).</li>
		</ol>
		</li>
		<li>Repeat this process for each site.</li>
		<li>Record counts in a lab notebook and on the computer for future analysis. Go to the menu bar, click on &#34;File&#34;, &#34;Save Data File&#34; to save the tracing and counts.</li>
	</ol>
	</li>
	<li>Ensure that inter-rater reliability (using correlation of counts) of the two uninformed raters for each section in each ROI always exceeds 0.8.</li>
</ol>
6. Statistics
<ol>
	<li>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.</li>
	<li>Compare saccharin intake (Day 4) with test intakes (Day 5) of the six groups using a randomized-block 2-way ANOVA69.</li>
	<li>Use Tukey comparisons (p &#60; 0.05) to determine individual significant effects69.</li>
	<li>Determine inter-rater reliability, and then use a common observer&#39;s counts.</li>
	<li>Average c-fos counts for the three representative slices for each site69.
	<ol>
		<li>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.</li>
		<li>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 &#60; 0.05) to reveal individual significant effects69.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Establish whether significant relationships between solution intakes and c-fos activation in each of the sites were observed using Bonferroni r correlations (p &#60; 0.05).
	<ol>
		<li>Systematically compare c-fos counts in the perilimbic and infralimbic pre-frontal cortex for each animal in the 3.5% corn oil group.</li>
		<li>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.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>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 &#60; 0.05).</li>
</ol>

The authors have no competing financial interests.

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...

Brain dopamine (DA) has been implicated in central responses to intake of palatable sugars through proposed hedonic1,2, effort-related3 and habit-based4,5 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 reviews6,7). The VTA has been implicated in sucrose intake8,9, and DA release is observed following sugar intake in the NAC10-15, AMY16,17 and mPFC18-20. 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 feeding22,23. Kelley24-27 proposed that these multiple projection zones of this DA-mediated system formed an integrated and interactive distributed brain network through extensive and intimate interconnections28-34.
In addition to the ability of DA D1 and D2 receptor antagonists to reduce intake of sugars35-37 and fats38-40, 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 mPFC47-49 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 AMY51,52.
The c-fos technique53,54 has been employed to investigate neural activation induced by palatable intake and neural activation. The term &#34;c-fos activation&#34; 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 NAC55-57. 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 AMY59,60. 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 sites65-67.
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 networks24-27. 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 network24-27. 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.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sprague-Dawley rats</td><td>Charles River Laboratories</td><td>CD-1</td><td></td></tr><tr><td>Wire Mesh Cages</td><td>Lab Products, Seaford, DE</td><td>30-Cage rack</td><td></td></tr><tr><td>Rat Chow</td><td>PMI Nutrition International</td><td>5001</td><td></td></tr><tr><td>Taut Metal Spring</td><td>Lab Products, Seaford, DE</td><td>n/a</td><td></td></tr><tr><td>Rat Weighing Scale</td><td>Fisher Scientific Company</td><td>n/a</td><td></td></tr><tr><td>Nalgene Centrifuge Tubes</td><td>Lab Products, Seaford, DE</td><td>10-0501</td><td></td></tr><tr><td>Rubber Stopper</td><td>Lab Products, Seaford, DE</td><td>n/a</td><td></td></tr><tr><td>Metal Sippers</td><td>Lab Products, Seaford, DE</td><td>n/a</td><td></td></tr><tr><td>Saccharin</td><td>Sigma Chemical Co</td><td>82385-42-0</td><td></td></tr><tr><td>Kool-Aid, Cherry</td><td>Kool-Aid</td><td>Commerical</td><td></td></tr><tr><td>Kool-Aid, Grape</td><td>Kool-Aid</td><td>Commercial</td><td></td></tr><tr><td>Fructose</td><td>Sigma Chemical Co</td><td>F0127</td><td></td></tr><tr><td>Glucose</td><td>Sigma Chemical Co</td><td>G8270</td><td></td></tr><tr><td>Corn Oil</td><td>Mazzola</td><td>Commerical</td><td></td></tr><tr><td>Xanthan Gum</td><td>Sigma Chemical Co</td><td>11138-66-2</td><td></td></tr><tr><td>Sliding Microtome</td><td>Microm International</td><td>n/a</td><td></td></tr><tr><td>Neurolucida Camera</td><td>MBF Bioscience</td><td>Software application</td><td></td></tr><tr><td>Gelatin-coated Slides</td><td>Fisher Scientific Company</td><td>12-550-343</td><td></td></tr><tr><td>Cover glass</td><td>Fisher Scientific Company</td><td>12-545-M</td><td></td></tr><tr><td>Golden Nylon Brushes</td><td>Loew-Cornell&amp;nbsp;</td><td>2037</td><td></td></tr><tr><td>Natural Hair Sable&amp;nbsp;</td><td>Loew-Cornell&amp;nbsp;</td><td>2022</td><td></td></tr><tr><td>24 Well Plates</td><td>Fisher Scientific</td><td>3527</td><td></td></tr><tr><td>6 Well Plates</td><td>Fisher Scientific</td><td>3506</td><td></td></tr><tr><td>1 L Pyrex bottles</td><td>Fisher Scientific</td><td>1395-1L</td><td></td></tr><tr><td>Tissue insert (tissue strainer)</td><td>Fisher Scientific</td><td>7200214</td><td></td></tr><tr><td>Eagle pipettes&amp;nbsp;</td><td>World Precision Instruments</td><td>E10 for 1-10&amp;#956;l</td><td></td></tr><tr><td>Eagle pipettes&amp;nbsp;</td><td>World Precision Instruments</td><td>E100 for 20-100&amp;#956;l</td><td></td></tr><tr><td>Eagle pipettes&amp;nbsp;</td><td>World Precision Instruments</td><td>E200 for 50-200&amp;#956;l</td><td></td></tr><tr><td>Eagle pipettes&amp;nbsp;</td><td>World Precision Instruments</td><td>E1000 for 100-1000&amp;#956;l</td><td></td></tr><tr><td>Eagle pipettes&amp;nbsp;</td><td>World Precision Instruments</td><td>E5000 for 1000-5000&amp;#956;l&amp;nbsp;</td><td></td></tr><tr><td>Universal Tips .1-10 &amp;#956;l</td><td>World Precision Instruments</td><td>500192</td><td></td></tr><tr><td>Universal Tips 5-200 &amp;#956;l</td><td>World Precision Instruments</td><td>500194</td><td></td></tr><tr><td>Universal Tips 500-5,000 &amp;#956;l</td><td>World Precision Instruments</td><td>500198</td><td></td></tr><tr><td>Blade Vibroslice 100</td><td>World Precision Instruments</td><td>BLADE</td><td></td></tr><tr><td>DPX Mounting Medium&amp;nbsp;</td><td>Electron Microscopy&amp;nbsp;</td><td>13510</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>Biologix Research Co.</td><td>10-0501</td><td></td></tr><tr><td>Slide Boxes</td><td>Biologix Research Co.</td><td>41-6100</td><td></td></tr><tr><td>Orbital Shaker&amp;nbsp;</td><td>Madell Corporation&amp;nbsp;&amp;nbsp;</td><td>ZD-9556</td><td></td></tr><tr><td>weigh boats&amp;nbsp;</td><td>Fisher Scientific</td><td>02-202-100</td><td></td></tr><tr><td>5 ml disposable pipettes</td><td>Fisher Scientific</td><td>13-711-5AM</td><td></td></tr><tr><td>Stereo Investigator Software</td><td>Micro Bright Field</td><td>Software application</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde Granular</td><td>Fisher Scientific</td><td>19210</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271-1</td><td></td></tr><tr><td>Sodium Phophate Monobasic</td><td>Fisher Scientific</td><td>S468-500</td><td></td></tr><tr><td>Sodium Phosphate Diphasic</td><td>Fisher Scientific</td><td>BP332-500</td><td></td></tr><tr><td>Hydrogen Peroxide&amp;nbsp;</td><td>Fisher Scientific</td><td>H324-500</td><td></td></tr><tr><td>SafeClear II&amp;nbsp;</td><td>Fisher Scientific</td><td>23-044-192</td><td></td></tr><tr><td>Methanol&amp;nbsp;</td><td>Fisher Scientific</td><td>A412-1</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Vector</td><td>S-1000</td><td></td></tr><tr><td>Biotinylated Anti-Rabbit IgG (H+L)</td><td>Vector</td><td>BA-1000</td><td></td></tr><tr><td>ABC Kit Peroxidase Standard</td><td>Vector</td><td>PK-4000</td><td></td></tr><tr><td>Anti-cFos (Ab-5) Rabbit</td><td>EMD chem/Cal Biochem</td><td>PC38</td><td></td></tr><tr><td>Triton X 100</td><td>SigmaAldrich</td><td>X-100</td><td></td></tr><tr><td>3,3&#039; diaminobenzidine tetra hydrochloride&amp;nbsp;</td><td>SigmaAldrich</td><td>D5905</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>SigmaAldrich</td><td>5881</td><td></td></tr><tr><td>Primary TH anti body</td><td>EMD Millipore</td><td>AB152</td><td></td></tr><tr><td>Euthosol</td><td>Virbac AH</td><td></td><td></td></tr></tbody></table>

simultaneous detection of c-fos activation from mesolimbic and mesocortical dopamine reward sites following naive sugar and fat ingestion in rats

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.

All representative results described below have been published previously69, and are re-presented here to support &#34;proof of concept&#34; 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 &#60; 0.001) with intakes (Day 1: 1.3 (&#177; 0.2) ml; Day 2...

Watch this Scientific Journal Video about Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats at JoVE.com

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

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.

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 ...

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9.0K Views.  CUNY, New York, NY.  Het doel van deze studie is om op beloning van gedistribueerde hersenprocessen netwerken te identificeren door het afbakenen van een betrouwbare immunohistologische techniek met behulp van mobiele c-fos activering gelijktijdige veranderingen in de dopamine wegen en terminal plaatsen na roman inname van vet en suiker in ratten te meten. 

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

A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste reactivity with the behavioral aspects of operant self-administration of drugs. Under very brief general anaesthesia, rats are implanted with an intraoral (IO) cannula that allows delivery of test solutions directly in the oral cavity. Animals are then tested in operant self-administration chambers whereby they can press a lever to receive IO infusions of test solutions. IO self-administration has several advantages over experimental procedures that involve drinking a solution from a spout&#160;or operant responding for solid pellets or solutions delivered in a receptacle. Here, we show that IO self-administration can be employed to study self-administration of high fructose corn syrup (HFCS). Rats were first tested for self-administration on a progressive ratio (PR) schedule, which assesses the maximum amount of operant behavior that will be emitted for different concentrations of HFCS (i.e.&#160;8%, 25%, and 50%). Following this test, rats self-administered these concentrations on a continuous schedule of reinforcement (i.e.&#160;one infusion for each lever press) for 10 consecutive days (1 session/day; each lasting 3 hr), and then they were retested on the PR schedule. On the continuous reinforcement schedule, rats took fewer infusions of higher concentrations, although the lowest concentration of HFCS (8%) maintained more variable self-administration. Furthermore, the PR tests revealed that 8% had lower reinforcing value than 25% and 50%. These results indicate that IO self-administration can be employed to study acquisition and maintenance of responding for sweet solutions. The sensitivity of the operant response to differences in concentration and schedule of reinforcement makes IO self-administration an ideal procedure to investigate the neurobiology of voluntary intake of sweets.

The current study evaluates a novel procedure for assessing the reinforcing effects of palatable solutions in laboratory rats: intraoral self-administration. To this end, operant responding (i.e.&#160;lever pressing) for intraoral infusions of sweet solutions at different concentrations was measured on continuous and progressive ratios schedules of reinforcement.

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste ...

Behavior

Studying Food Reward and Motivation in Humans

A key challenge in studying reward processing in humans is to go beyond subjective self-report measures and quantify different aspects of reward such as hedonics, motivation, and goal value in more objective ways. This is particularly relevant for the understanding of overeating and obesity as well as their potential treatments. In this paper are described a set of measures of food-related motivation using handgrip force as a motivational measure. These methods can be used to examine changes in food related motivation with metabolic (satiety) and pharmacological manipulations and can be used to evaluate interventions targeted at overeating and obesity. However to understand food-related decision making in the complex food environment it is essential to be able to ascertain the reward goal values that guide the decisions and behavioral choices that people make. These values are hidden but it is possible to ascertain them more objectively using metrics such as the willingness to pay and a method for this is described. Both these sets of methods provide quantitative measures of motivation and goal value that can be compared within and between individuals.

This article describes a set of methods for the measurement of food related motivation and food related goal values in humans.

A key challenge in studying reward processing in humans is to go beyond subjective self-report measures and quantify different aspects of reward such as ...

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats

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 &#8211; bypassing reward-directed behavior and voluntary drinking behavior &#8211; allowing for direct examination of DA responses to tastant stimuli.

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.

Rapid, phasic dopamine (DA) release in the mammalian brain plays a critical role in reward processing, reinforcement learning, and motivational control. ...

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can produce a rewarding effect that strengthens action-outcome associations and reinforces future behavior directed at obtaining these foods. Increasing evidence that the rewarding effects of energy-dense foods play a profound role in overeating and the development of obesity has heightened interest in studying the genes, molecules and neural circuitry that modulate food reward1,2. The rewarding impact of different stimuli can be studied by measuring the willingness to work to obtain them, such as in operant conditioning tasks3. Operant models of food reward measure acquired and voluntary behavioral responses that are directed at obtaining food. A commonly used measure of reward strength is an operant procedure known as the progressive ratio (PR) schedule of reinforcement.4,5 In the PR task, the subject is required to make an increasing number of operant responses for each successive reward. The pioneering study of Hodos (1961) demonstrated that the number of responses made to obtain the last reward, termed the breakpoint, serves as an index of reward strength4. While operant procedures that measure changes in response rate alone cannot separate changes in reward strength from alterations in performance capacity, the breakpoint derived from the PR schedule is a well-validated measure of the rewarding effects of food. The PR task has been used extensively to assess the rewarding impact of drugs of abuse and food in rats (e.g.,6-8), but to a lesser extent in mice9. The increased use of genetically engineered mice and diet-induced obese mouse models has heightened demands for behavioral measures of food reward in mice. In the present article we detail the materials and procedures used to train mice to respond (lever-press) for a high-fat and high-sugar food pellets on a PR schedule of reinforcement. We show that breakpoint response thresholds increase following acute food deprivation and decrease with peripheral administration of the anorectic hormone leptin and thereby validate the use of this food-operant paradigm in mice.

The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Medicine

Palatable Western-style Cafeteria Diet as a Reliable Method for Modeling Diet-induced Obesity in Rodents

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases.&#160;The health burden of obesity and its comorbid conditions highlight the need for better understanding of its pathogenesis, yet ethical constraints limit studies in humans. To this end externally valid models of obesity in laboratory animals are essential for the understanding of being overweight and obesity.&#160;While many species have been used to model the range of changes that accompany obesity in humans, rodents are most commonly used. Our laboratory has developed a western-style cafeteria diet that consistently leads to considerable weight gain and markers of metabolic disease in rodents. The diet exposes rodents to a variety of highly palatable foods to induce hyperphagia, modeling the modern western food environment. This diet rapidly induces weight gain and body fat accumulation in rats allowing for the study of effects of overeating and obesity. While the cafeteria diet may not provide the same control over macronutrient and micronutrient profile as purified high-fat or high-fat, high-sugar diets, the cafeteria diet typically induces a more severe metabolic phenotype than that observed with purified diets and is more in line with metabolic disturbances observed in the overweight and obese human population.

This protocol describes the use of a highly palatable, western-style cafeteria diet to model overeating and obesity in rodents. Here, we provide a detailed outline of food selection, preparation and measurement, and explain methodological factors that assist in generating a robust and reproducible phenotype.

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases.&#160;The health ...

Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral studies in animal models. In this protocol, to study depression-like and anhedonic behavior in rats, the established sucrose preference and novelty-induced hypophagia tests are combined with an automated food and liquid intake monitoring system. Prior to testing, in the sucrose preference paradigm, male rats are trained for at least 2 days to consume a sucrose solution in addition to tap water. During the test, rats are again exposed to water and sucrose solution. Consumption is registered every second by the automated system. The ratio of sucrose to total water intake (sucrose preference ratio) is a surrogate parameter for anhedonia. In the novelty-induced hypophagia test, male rats undergo a training period in which they are exposed to a palatable snack. During training, rodents show a stable baseline snack intake. On test day, the animals are transferred from home cages into a fresh, empty cage representing a novel unknown environment with access to the known palatable snack. The automated system records the total intake and its underlying microstructure (e.g., latency to approaching the snack), providing insight into anhedonic and anxious behaviors. The combination of these paradigms with an automated measuring system provides more detailed information, along with higher accuracy by reducing measuring errors. However, the tests use surrogate parameters and only depict depression and anhedonia in an indirect manner.

Presented here is a protocol to study depression-like and anhedonic behavior in rats. It combines two well-established behavioral methods, the sucrose preference and novelty-induced hypophagia tests, with an automated food and liquid intake monitoring system, to indirectly investigate rodent behavior using surrogate parameters.

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

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

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