Name: simultaneous detection of c-fos activation from mesolimbic and mesocortical dopamine reward sites following naive sugar and fat ingestion in rats
Uploaded: 2016-08-24T13:00:00
Description: 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

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 samp...

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.

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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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