Acute High Fat Diet Consumption Activates the Mesolimbic Circuit and Requires Orexin Signaling in a Mouse Model

Overconsumption of palatable energy-dense foods has negative health implications and it is associated with obesity and several eating disorders. Currently, little is known about the neuronal circuitries activated by the acute ingestion of a rewarding stimulus. Here, we used a combination of immunohistochemistry, pharmacology and neuronal tracing analyses to examine the role of the mesolimbic system in general, and the orexin neurons in particular, in a simple experimental test in which naïve mice are allowed to spontaneously eat a pellet of a high fat diet (HFD) for 2 h. We found that acute HFD activates c-Fos expression in several reward-related brain areas, including the ventral tegmental area (VTA), nucleus accumbens, central amygdala and lateral hypothalamic area. We also found that: i- HFD-mediated orosensory stimulation was required for the mesolimbic pathway activation, ii- acute HFD differentially activates dopamine neurons of the paranigral, parabrachial pigmented and interfascicular sub-regions of the VTA, and iii- orexin neurons of the lateral hypothalamic area are responsive to acute HFD. Moreover, orexin signaling blockade, with the orexin 1 receptor antagonist SB-334867, reduces acute HFD consumption and c-Fos induction in the VTA but not in the other mesolimbic nuclei under study. Finally, we found that most orexin neurons responsive to acute HFD innervate the VTA. Our results show that acute HFD consumption recruits the mesolimbic system and that the full manifestation of this eating behavior requires the activation of orexin signaling.


Introduction
Consumption of palatable energy-dense food is a rewarding experience for most animals, including human beings. An emerging literature suggests that the current environment, where inexpensive palatable foods are easily accessible, promotes overconsumption of calories that can lead to obesity or eating disorders [1][2][3][4]. In terms of the neurobiological bases of this phenomenon, some evidence suggests that the hedonic brain circuits that drive consumption based on the rewarding properties of foods can override brain circuits that drive food intake depending on energy store levels, leading to intake of calories beyond energetic and nutritional requirements [1][2][3][4]. However, the neuronal circuitries activated by the acute ingestion of a rewarding stimulus are currently unclear. The mesolimbic system participates in the reinforcing and motivational effects of several rewarding stimuli [5]. This system consists of dopamine neurons that project from the ventral tegmental area (VTA) to various forebrain areas including the accumbens (NAc) as well as the central amygdala (CeA), prefrontal cortex, hippocampus and hypothalamus [6,7]. The VTA is a neuroanatomically and functionally complex brain area that contains diverse neuronal populations that may play distinct roles in reward-related behaviors [8,9]. It is currently unclear whether the mesolimbic system plays a role in the hedonic-driven food consumption [7,10,11]. The mesolimbic system is activated in response to palatable foods, and dopamine release in the NAc augments the drive to obtain food rewards [12][13][14]. However, NAc dopamine depletion alone does not alter feeding, and pharmacological blockade of dopamine receptors in the NAc affects motor behavior and has no effects on food intake [13,15]. The NAc shell has been implicated in hedonic eating since it sends projections to neurons of the lateral hypothalamic area (LHA) controlling food intake [12,16]. In the LHA, orexin (also known as hypocretin)producing neurons seem to be under a tonic inhibition that can be relieved by activation of reward pathways [16][17][18]. LHA orexin neurons regulate the VTA dopamine neurons and have been implicated in food reward modulation [17][18][19][20][21][22][23]. To our knowledge, it has not been explored whether the VTA-NAc-LHA pathway plays a role in acute consumption of a highly palatable food. Here, we used a combination of immunohistochemistry, pharmacology and neuronal tracing analyses to examine the role of the neuronal populations of the VTA-NAc-LHA pathway during acute HFD consumption.

Animals and diets
Adult (2-3 month old) C57BL6/J mice were generated at the animal care facility of the Multidisciplinary Institute of Cell Biology (IMBICE). Male mice were housed under a 12-h light/ dark cycle (lights on at 07:00 a.m.) and with regular chow and water available ad lib. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council, USA [24], and all efforts were made to minimize suffering. The protocol was approved by the Institutional Animal Care and Use Committee of the IMBICE (approval ID 10-0113).

Experimental protocols
In the morning of the experimental day (9.00 a.m.), ad lib fed mice were exposed to a pellet of either RC or HFD for 2 h (n = 16 and 14, respectively). In order to distinguish potential effects of the HFD itself and those of higher food intake, a pair-fed control group consisting of mice exposed to amounts of HFD similar to those eaten by mice exposed to RC was included (n = 10). The cumulative food intake was recorded 2 h after food exposure.
In order to perform acute intragastric feeding, either RC or HFD were resuspended in water and administered via plastic feeding tubes for oral rodent gavage (cat# FTP-20-38, Instech Solomon Laboratories). Oral gavage procedure was performed by trained personnel as we have previously done in the past [25]. The distance from the tip of the animal's head to the last rib was used as a reference for the length of the gavage tube in order to avoid stomach perforation. Mice were made accustomed to handling and trained on the procedure daily for at least 4 days prior to experimentation to reduce stress. Briefly, the mouse's head was extended backwards to create a straight line through the neck and esophagus. The gavage tube was placed in the diastema of the mouth and then gently advanced along the upper palate until the end of the esophagus was reached. Mice (n = 4 per group) were given one infusion intragastrically containing 310 mg of diet as a slurry in a total volume of 1 mL. Of note, control group received ,0.77 kcal and HFD group received ,1.21 kcal. After infusion, the tube was gently removed following the same angle as insertion. Then, mice were returned the home cage and anesthetized 2 h later.
In another experiment, orexin 1 receptor antagonist SB-334867 (Tocris, cat.# 1960) was used to block orexin signaling. This compound was i.p. injected at a dose of 5 mg/g body weight in 200 mL saline at 8.30 a.m. A similar dose of SB-334867 has been shown to affect high-fat intake in many models while it does not affect food intake of freely-available RC [25][26][27][28]. Thirty min after treatment, ad lib fed mice injected with either vehicle or SB-334867 were exposed to a pellet of HFD for 2 h (n = 10 and 8, respectively). Here, an extra pair-fed control group consisting of vehicle-injected mice exposed to amounts of HFD similar to those eaten by SB-334867-treated mice was included in order to distinguish the specific effects of SB-334867 (n = 9). The cumulative food intake was recorded 2 h after food exposure.

Tracing Studies
In order to label LHA neurons innervating the VTA, the retrograde tracer FluoSpheres (Red Fluorescent, Invitrogen cat.# F8793) was stereotaxically microinjected into the VTA (n = 6). The placement coordinates were: AP -3.52, L 0.42, and V 4.27 mm to bregma. These coordinates were initially obtained from the Paxinos atlas [29] and then modified based on pilot studies. The injector was positioned with a micromanipulator and an 800 nL volume of 2.5% FluoSpheres in saline was injected via Effects of High Fat Diet on the Mesolimbic Pathway PLOS ONE | www.plosone.org a 33-gauge injector. The injector was left in place for 10 min following the injection to allow the tracer to diffuse away from the injection site. After 6 days for retrograde transport of the FluoSpheres, ad lib fed mice were exposed to a pellet of HFD for 2 h and then anesthetized. Control group was excluded from this experiment because this study was specifically performed to test if orexin neurons responsive to acute HFD, as indicated by the presence of c-Fos signal, send their projections to the VTA. Of note, stereotaxic microinjections of FluoSpheres in the VTA resulted in 3 missed injections in which the tracer did not diffuse to all three VTA subdivisions. These mice were excluded from the analysis.

C-Fos immunostaining
Two hour after spontaneous food intake or acute intragastric feeding, mice were anesthetized and perfused with formalin as previously described [30]. Then, brains were removed, immersed in 20% sucrose overnight and coronally cut at 20 mm into three equal series on a sliding cryostat. C-Fos immunostaining was performed as described before [30]. Briefly, sections were pretreated with H 2 O 2 , treated with blocking solution and incubated with anti-c-Fos antibody (Calbiochem/Oncogene, cat# PC38, 1:10,000) for 2 days at 4uC. Then, sections were incubated with biotinylated donkey anti-rabbit antibody (Jackson ImmunoResearch Laboratories, cat# 711-065-152, 1:1,000) for 1 h, and with Vectastain Elite ABC kit (Vector Laboratories, cat# PK-6200) for 1 h, according to manufacturer's protocols. Then, visible signal was developed with 3-39-diaminobenzidine (DAB)/Nickel solution (Sigma Aldrich, cat# 32750), which generated a black/ purple precipitate. Sections were sequentially mounted on glass slides and coverslipped with mounting media. Bright-field images were acquired with a Nikon Eclipse 50i and a DS-Ri1 Nikon digital camera. Adobe Photoshop CS2 image editing software was used to adjust levels, contrast and brightness.

Double c-Fos and tyrosine hydroxylase (TH) immunostaining
Independent series of brain sections were used for double immunostaining for c-Fos and TH (n = 6 per group). After c-Fos immunostaining was completed, brain sections were incubated 48 h with an anti-TH antibody (Sigma-Aldrich, cat# T2928, 1:4,000) and then sequentially incubated with biotinylated donkey anti-mouse antibody (Vector Laboratories, cat# BA-9200, 1:2,000) and Vectastain Elite ABC kit as detailed above. Finally, visible signal was developed by incubation with DAB solution, showing a brown precipitate. Sections were sequentially mounted on glass slides and coverslipped with mounting media. Bright-field images were obtained as described above.

Double c-Fos and orexin immunostaining
Orexin immunostaining was performed using an anti-orexin antibody (Phoenix Pharmaceuticals, cat# H-003-30, 1:20,000). In one experiment, c-Fos immunostained brain sections were used to detect orexin-IR signal using brown chromogenic immunostaining as indicated above (n = 6 per group). In samples from tracing studies, c-Fos immunostained brain sections were used for detection of orexin-IR signal using fluorescent immunostaining. After washing, brain slices were incubated overnight with the antiorexin antibody. Next day, sections were incubated with fluorescent donkey anti-rabbit Alexa 488 antibody (Invitrogen, cat# A21207, 1:1,000) during 2 h, mounted, and coverslipped in a fluorescence mounting solution-containing DAPI. Results were visualized using either fluorescence (orexin and FluoSpheres) or bright-field light (c-Fos) sources and images were obtained as described above.
Single orexin and double TH/orexin immunostaining within the VTA Brain sections containing the VTA were used for detection of orexin-IR signal using the anti-orexin antibody and black/purple chromogenic immunostaining as indicated above (n = 6 per group). Independent series of brain sections were used first for detection of TH-IR signal using the anti-TH antibody and brown chromogenic immunostaining, and then for orexin immunostaining using the anti-orexin antibody and black/purple chromogenic immunostaining as indicated above (n = 6 per group).

Quantitative neuroanatomical analysis
To determine the total number c-Fos-immunoreactive (IR) cells in each brain region, cells containing distinct nuclear black/purple precipitate were quantified in one out of three complete series of coronal brain sections. Anatomical limits of each brain region were identified using a mouse brain atlas [29]. Total c-Fos-IR cells in each brain region were estimated in sections between bregma 0.86 and 1.54 mm for the NAc; between bregma 20.70 and 21.70 mm for the CeA and between bregma 20.82 and 21.94 mm for the LHA. For the analysis, the VTA was subdivided into paranigral nucleus (PN), parabrachial pigmented area (PBP) and interfascicular nucleus (IF), according to previous descriptions, and analyzed between bregma 23.08 and 23.80 mm [8,31,32]. Data were expressed as c-Fos-IR cells per coronal section. Blind quantitative analysis was performed independently by two observers, in 10-14 animals per experimental condition. In the case of c-Fos/TH co-staining, TH-IR was confined to the perikarya and dendrites, thus allowing visualization of the nucleus with or without black/purple label for c-Fos. Total TH-IR neurons and TH-IR neurons with nuclei positive for c-Fos were counted in the different VTA sub-regions. Orexin-IR was also confined to the perikarya and dendrites, which allowed visualization of the nuclei with or without black/purple label for c-Fos. Total orexin-IR neurons and orexin-IR neurons with nuclei positive for c-Fos were counted in each side of the third ventricle. Then, double immunostaining analyses were expressed as the total number of neurons positive for c-Fos observed in each brain region. The estimation of total TH-IR cells in the VTA and total orexin-IR cells in the LHA were corrected for double counting, according to the method of Abercrombie [33], where the ratio of the actual number of neurons to the observed number is represented by T/T+h where T = section thickness, and h = the mean diameter of the neuron. For this, cell diameter was quantified, of at least 40 cells in each brain area and experimental group, using the software ImageJ.
For quantitative estimates of orexin-IR fibers density within each VTA sub-region, images of VTA sections labeled for orexin were acquired with a 100X objective lens, in comparable areas and under the same optical and light conditions. To estimate the orexin-IR fiber density, microphotographs were transformed to 8bit greyscale images, and the mean optical density (OD) for each image was measured using ImageJ software. The OD measurement for each image is the mean grey value of the pixels, with a 256 grayscale value as a reference. Nonspecific background was determined in the dorsal midbrain, where no orexin-IR fibers were detected. The OD value for each VTA sub-region corresponds to the mean orexin-IR fibers density minus the respective background level of the sample. For quantitative estimation of the number of somata of TH-IR neurons contacted by orexin-IR fibers (axonal boutons), sections double labeled for TH and orexin were visualized using a 100X objective lens and images were acquired as described above. Then, total TH-IR neurons and TH-IR neurons contacted by orexin-IR fibers were counted per high magnification picture and results were expressed as total TH-IR neurons contacted by orexin-IR fibers in each VTA sub-region. In tracing studies, triple-, double-and single-labeled neurons were counted, and relationships were expressed as percentage of either double-or single-labeled cells.

Statistical analyses
Data were expressed as mean6SEM. One-way ANOVA followed by the Newman Keuls test was used to compare food intake or quantitative analysis of neuroanatomical data. Correlation between 2 h food intake and c-Fos-IR cells in the IF subregion of the VTA was performed by linear regression analysis using a general linear model. Significant differences were considered when p,0.05.

Acute HFD activates c-Fos expression in reward-related brain areas
In order to map brain areas responsive to acute HFD, ad-lib fed mice were exposed to a pellet of either RC or HFD for 2 h and then processed for immunostaining. Total food intake was significantly higher in animals exposed to HFD as compared to those exposed to RC (311635 vs. 110623 mg respectively, p,0.001, Figure 1B). Total food intake in HFD pair-fed mice was 119615 mg. Since RC and HFD were not isocaloric, a significant difference of caloric intake between the experimental groups was also observed [F(2,34) = 22.64, P,0.0001]. Total caloric intake for control, HFD-ad lib and HFD-pair-fed groups was 275667, 12166146 and 455656 cal, respectively ( Figure 1C). Thus, caloric intake in HFD-ad lib mice remained significantly higher as compared to caloric intake observed in both control and HFDpair-fed groups (P,0.001) while caloric intake did not differ between control and HFD-pair-fed groups (P.0.05). The density of observed c-Fos-IR cells is outlined in Table 1, which also includes the abbreviations used in the figures and throughout the text. C-Fos-IR signal within the hypothalamus was moderate and enriched in some nuclei such as the PVH, DMN and LHA. However, the higher number and strongest intensity of c-Fos-IR cells were observed in several nuclei of the mesolimbic pathway, including the VTA, NAc and CeA. Thus, a detailed quantitative analysis was performed in these areas. Acute  Figure 3C). In the CeA, 9.362.2, 42.266.7 and 35.267.1 c-Fos-IR cells were detected in control, HFD-ad lib and HFD-pair-fed groups, respectively (P,0.01 vs. control group, Figure 3D). In the LHA, 4.864.1, 57.768.3 and 61.368.1 c-Fos-IR cells were detected in control, HFD-ad lib and HFD-pair-fed groups, respectively (P,0.001 vs. control group, Figure 3E). Thus, these data indicate that acute HFD activates neuronal populations of the mesolimbic pathway.
Acute gastric tube feeding with HFD fails to activate c-Fos expression in the mesolimbic pathway

Acute HFD activates c-Fos expression in TH-IR cells of VTA
To determine if dopamine neurons of the VTA were activated by acute HFD consumption, double immunohistochemistry for c-Fos/TH was performed ( Figure 4). Cytoarchitectonic features of TH-IR cells within the different sub-regions of the VTA were similar to those described for the rat [8]. TH-IR cells in the PN were relatively homogeneous, mostly medium sized, medium to dark stained and semi-laterally oriented. TH-IR cells in the PBP were large, medium to low stained and with no uniform orientation. The IF is localized just dorsal to the interpeduncular nucleus; TH-IR cells within this nucleus were small, medium to dark stained and densely packed along the midline.

Orexin signaling blockade reduces acute HFD consumption and c-Fos induction in the VTA
To determine if orexin signaling was required for acute HFD consumption, the orexin receptor 1 selective antagonist SB-334867 was administrated to ad lib fed mice that were subsequently exposed to HFD. SB-334867 treatment reduced food intake (212627 vs. 119611 mg, p,0.01 vs. vehicle-treated group), and, as a consequence, an extra pair-fed group was added to the experiment ( Figure 6

LHA orexin neurons responsive to acute HFD innervate the VTA
In order to identify a neuroanatomical substrate of orexin signaling within the VTA, the orexin fibers within all three subregions of this nucleus were quantified. Orexin-IR terminals with characteristic bouton morphology were observed in all three subregions of the VTA ( Figure 8A). The relative density of orexin-IR fibers in the PN, PBP and IF sub-regions of the VTA was 46.364.2, 48.266.4 and 43.662.2 OD/100X field, respectively (p.0.05). In order to determine if orexin signaling differentially affected TH-IR neurons in the VTA sub-regions, we performed TH and orexin co-staining. Orexin-IR terminals making close contacts with TH-IR neurons were found in the three sub-regions of the VTA ( Figure 8B). Quantitative analysis indicated that 84.668.8, 86.766.6 and 95.968.7 TH-IR neurons had apparent contacts by orexin-IR fibers in the PN, PBP and IF sub-regions of the VTA, respectively (p.0.05). Apparent appositions were also observed in TH-IR dendrites.
Finally, we tested if orexin neurons sending their projections to the VTA were responsive to acute HFD. For this purpose, mice were subjected to red FluoSpheres injection in the VTA, exposed to HFD and then used for further double c-Fos and orexin

Conclusions
In the current study we provide evidence that acute HFD consumption recruits centers of the mesolimbic pathway including neurons of the VTA, NAc, CeA and LHA. Remarkably, we found different responsiveness of PBP, PN and IF sub-regions of the VTA to acute HFD consumption. In terms of the neuronal circuit recruited by acute HFD, we show that orexin signaling is necessary for the spontaneous consumption of HFD and for the HFDinduced activation of the mesolimbic dopamine system. In support of this possibility, we show that HFD-responsive orexin neurons of the LHA innervate the VTA.
Current findings support the notion that brain circuits driving motivation to consume a palatable food are a powerful system that can override homeostatic signals [4]. In our experimental test, mice exposed to a HFD pellet spontaneously consumed ,1.22 kcal, which represents nearly a fifth of their daily food intake, in a short period of time. Of note, satiated mice were exposed to HFD at a time of the day when spontaneous food intake is minimal and while they remained with free access to RC. Thus, this spontaneous consumption of HFD appears to be mainly due to the palatable nature of the stimulus and involve hedonic aspects of eating. In support of this possibility, we observed a strong activation of different nuclei of the mesolimbic system.  Importantly, acute HFD resulted in the same profile of c-Fos activation in the mesolimbic pathway in mice that had been previously exposed 2 h to HFD in the previous day (data not shown). Thus, neophobia (i.e. aversion to approach a novel stimulus) or novelty seeking (i.e. enhanced specific exploration of a novel stimulus) seems to play a minor role in our observations. This could be due to the minimal differences in the overall appearance of the HFD and RC pellets. Also, HFD and RC pellets were prepared with the same ingredients though in different proportion. We cannot completely rule out that some degree of neophobia exists in our experimental paradigm. However, the large amount of food intake in HFD-ad lib group and the lack of activation of brain nuclei known to be activated in response to a novel gustatory stimulus [34] suggest that neophobia, if present at all, is transient and minimal. Thus, we propose that the current behavioral test is a suitable model to investigate the neuronal circuits and molecular mechanisms regulating hedonic-related eating behaviors.
It is interesting to stress that we included an experimental group consisting of mice exposed to an amount of HFD weight similar to that eaten by control mice exposed to RC. This HFD-pair-fed group was used to distinguish potential effects of the HFD itself from those of higher food intake. Although RC and HFD were not isocaloric, the amount of calories consumed by HFD-pair-fed and control mice were statistically indistinguishable. Thus, HFD-pairfed and control mice consumed similar amounts of food, expressed either as food weight or calories. HFD ad lib and HFD-pair-fed groups showed similar acute HFD-induced activation of most  neuronal populations of the mesolimbic pathway, with the exception of the IF neurons of the VTA that were not activated in the HFD-pair-fed group (see below). Thus, it seems that the acute activation of the mesolimbic pathway is mainly mediated by the HFD itself, rather than the eaten weight or the caloric content.
We show that HFD-mediated orosensory stimulation is required for the mesolimbic pathway activation since intragastric administration of HFD failed to increase c-Fos in most of the brain areas under study. Recent studies suggest that dietary fat sensing would involve fatty acid receptors located in the tongue [35]. Then, orosensory taste information is transmitted to the NTS, which directly or indirectly recruits neuronal nuclei in the hindbrain (i.e. PBN), midbrain (i.e. VTA) and forebrain (i.e. NAc and LHA) [36,37]. Thus, it is possible to hypothesize that the HFD-mediated orosensory stimulation due to the high levels of fatty acids present in the diet, results in the activation of neuronal circuits that end up  recruiting the mesolimbic pathway. Post-oral effects of HFD have been proposed to result in overconsumption because fat is less satiating than other macronutrients [38]. In addition, it has been shown that the rewarding value of HFD is determined by its postoral nutritive effects [39]. Current data stress the importance of the HFD-induced oral stimulation in the acute activation of the mesolimbic pathway, but it does not refute post-ingestion effects of HFD on its rewarding value. One intrinsic limitation of intragastric gavage is that it may cause unusual gastric distention and change the dynamics of nutrient entry to the intestine. In addition, stress associated to the animals handling may mask physiological responses to nutrients. However, this experimental strategy is a valuable tool to dissociate the impact of oral and postoral signals as demonstrated by previous studies showing that intragastric infusions of glucose can stimulate dopamine system, independently of oral stimulation [40,41].
Here we show that VTA dopamine neurons are significantly activated in HFD-exposed mice. The initial site of action for addictive drugs is known to be predominantly the VTA neurons of the mesolimbic circuit, which then influences many behaviors related to drug addiction [11]. In contrast, the role of VTA neurons in hedonic-driven food consumption is still a matter of debate [10,13,14]. It has been proposed that VTA dopamine neurons are responsive to pleasurable foods, or cues that predict it, and also able to promote behaviors directed towards food consumption [5,42]. VTA contains multiple anatomically and functionally diverse sub-regions [8,9,31,32,43]. Here we have analyzed c-Fos expression within the VTA sub-regions where dopamine neurons are enriched [8] and found a differential response to the spontaneous HFD consumption. In particular, acute HFD caused a significant activation of dopamine neurons of the PN and PBP sub-divisions independently of the amount of HFD ingested. In contrast, activation of dopamine and nondopamine neurons of the IF sub-division of the VTA occurred only in mice that over-consumed HFD and the degree of activation correlated with the amount of HFD ingested. The finding that HFD-pair-fed group showed no activation of IF neurons, regardless of their motivation for HFD eating, suggests that these neurons are responsive to the amount of HFD ingested rather than triggering its consumption. Importantly, sub-divisions of the VTA also show differences in terms of their projections and neuronal targets [8]: the PBP and PN dopamine neurons project to the ventrolateral striatum, including the lateral part of the NAc shell and NAc core, while the IF neurons selectively project to the medial part of the NAc shell. Thus, it is possible to hypothesize that sub-divisions of the VTA participate in diverse aspects of hedonic eating. Of note, sub-divisions of the VTA have been shown to respond differently to drugs of abuse, including opiates and alcohol [44][45][46][47].
Acute HFD leads to a strong activation of LHA orexin neurons that is required for the activation of VTA neurons and the full induction of food intake. The LHA is a key brain area that integrates gustatory and visceral information and then modulates different functions, including feeding [17][18][19]. LHA Orexin neurons are involved not only in the regulation of homeostatic food intake itself but also in food reward-related behaviors, including hedonic and motivational components of eating [17][18][19]25]. In this regard, LHA orexin neurons project to dopamine neurons of the VTA, where orexin receptors are highly expressed and orexin activates both dopamine and non-dopamine neurons [17][18][19][20][21][22]. Orexin signaling is relevant for hedonic eating, as suggested by the finding that orexin-1 receptor antagonism attenuates HFD consumption in rats subject to forced caloric satiation [28]. Interestingly, the hypophagic effect of orexin-1 receptor antagonism occurs exclusively on high fat diet intake and does not affect RC intake [25][26][27][28]. It has been hypothesized that orexin signaling selectively enhances potentiation of glutamatergic synaptic transmission in the VTA for highly salient appetitive reinforcers [27]. However, the reason of the specificity of orexin signaling regulating HFD intake is currently unclear. Here we show that spontaneous consumption of HFD was partially blocked in mice administered an orexin 1 receptor antagonist. Thus, we have not only confirmed previous studies but also shown that even spontaneous consumption of HFD in ad lib fed mice requires orexin signaling.
Here, we also show that orexin signaling blockade reduced c-Fos induction in the VTA suggesting that orexin neurons are involved in the activation of the mesolimbic pathway. Indeed, we show that most LHA orexin neurons projecting to the VTA increased c-Fos in response to acute HFD. Thus, we conclude that acute HFD may indirectly engage the dopamine neurons of the mesolimbic system by initially targeting orexin LHA neurons which, in turn, project to the VTA. The estimations of orexin-IR fibers density and number of TH-IR neurons contacted by orexin-IR fibers within each VTA sub-region showed no differences. Thus, the mechanisms mediating the differential activation of dopamine neurons of the IF sub-region of the VTA in response to acute HFD will require further studies. Interestingly, we also found VTA innervating neurons of the LHA responsive to acute HFD that were negative for orexin-IR. These LHA non-orexin neurons responsive to HFD may include melanin-concentrating hormoneproducing or also neurotensin-producing neurons, which have been shown to reinforce the consumption of calorically dense foods [48,49]. Of note, a potential involvement of orexin signaling in the VTA mediating the acute stimulation of HFD consumption does not rule out that other neuronal targets of the orexin neurons also participate in this mechanism [22]. Indeed, orexin neurons project to other centers of the mesolimbic pathway known to mediate motivated behaviors, such as the prefrontal cortex, where orexin receptors are highly expressed [50][51][52]. Future studies, using selectively blockade of orexin 1 receptors in these orexin targets, are required in order to elucidate the circuit activated by acute HFD consumption.
Acute HFD leads to an activation of NAc, independent of the orexin signaling. The NAc is strategically positioned to translate the affective assessment of food into feeding behaviors since it receives critical orosensory information and then projects to hypothalamic and midbrain areas that contribute to the motor expression of feeding [12,16,53]. The NAc contains two functionally different sub-regions: NAc core and NAc shell [18]. The NAc core projects to the basal ganglia pathways to influence voluntary motor functions while the NAc shell projects mainly to subcortical limbic regions, such as the LHA and VTA, and modulates hedonic behaviors [18]. The role of the NAc in feeding behaviors is complex [16,50,53]. It has been shown that stimulation of NAc shell neurons by m-opiod receptor agonists can stimulate HFD consumption by recruiting LHA orexin neurons that, in turn, would activate VTA neurons [16,50,53]. Here we show that acute HFD activated c-Fos expression in the NAc shell and that this effect was not blocked by an orexin receptor 1 antagonist. Thus, it is possible to hypothesize that NAc shell is one of the initial neuronal targets recruited by HFD consumption that then further engages other brain systems, including the LHA and VTA neurons. In support of this possibility, it has been shown that the hyperphagia observed in response to intra-NAc shell administration of the m-opiod receptor agonist DAMGO is blocked by temporary inactivation of the LHA and VTA [53]. Although not a central focus of the current study, we observed that acute HFD also activates c-Fos expression in the NAc core and CeA in an orexin-independent manner.
Here, we have used a simple experimental paradigm in mice to provide evidence that clarify our understanding of the brain neuronal circuits recruited by the acute and spontaneous HFD consumption. Hopefully, this information will be helpful to the future development of new strategies for the treatment of conditions in which overconsumption of highly palatable food is observed. For instance, this current protocol could be used to study the neurobiology of binge-eating episodes, which refer to specific events of uncontrollable overconsumption in which the motivation to eat is usually aimed to obtain palatable energy-dense foods [54][55][56]. Episodes of binge-eating are characteristic of patients suffering from several eating disorders including anorexia nervosa, bulimia nervosa and binge eating disorder [57][58][59][60]. In addition, binge-eating episodes are observed in obese patients and even in healthy people under specific circumstances, such as stress [61]. Despite its clinical relevance, finding appropriate animal models for studying binge eating episodes has been challenging due to the fact that its etiology in humans is currently unclear [62]. In order to trigger acute food overconsumption, many animal models of binge eating involve a previous period of imposed food restriction, which also affects brain circuits regulating homeostatic aspects of food intake [55,63,64]. However, some evidences suggest that binge-eating episodes mainly involve hedonic-related feeding circuits [65]. Here, we show that satiated mice that spontaneously consumed a large amount of HFD in a short period of time have a strong activation of the VTA-NAc-LHA pathway. Therefore, this experimental protocol may be helpful to study the eating episodes in early stages, when the mesolimbic pathway would presumably play a key role [66].