Pairing binge drinking and a high-fat diet in adolescence modulates the inflammatory effects of subsequent alcohol consumption in mice

International Journal of Molecular Sciences Article Pairing Binge Drinking and a High-Fat Diet in Adolescence Modulates the Inflammatory Effects of Subsequent Alcohol Consumption in Mice Macarena González-Portilla 1, Sandra Montagud-Romero 2, Francisco Navarrete 3,4 , Ani Gasparyan 3,4, Jorge Manzanares 3,4, JoséMiñarro 1,4 and Marta Rodríguez-Arias 1,4,*   Citation: González-Portilla, M.; Montagud-Romero, S.; Navarrete, F.; Gasparyan, A.; Manzanares, J.; Miñarro, J.; Rodríguez-Arias, M. Pairing Binge Drinking and a High-Fat Diet in Adolescence Modulates the Inflammatory Effects of Subsequent Alcohol Consumption in Mice. Int. J. Mol. Sci. 2021,22, 5279. https://doi.org/10.3390/ijms22105279 Academic Editor: Giuseppe Esposito Received: 9 April 2021 Accepted: 15 May 2021 Published: 17 May 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Psychobiology, Facultad de Psicología, Universitat de Valencia, Avda. Blasco Ibáñez 21, 46010 Valencia, Spain; [email protected] (M.G.-P.); [email protected] (J.M.) 2Department of Psychology and Sociology, University of Zaragoza, C/ Ciudad Escolar s/n, 44003 Teruel, Spain; [email protected] 3Instituto de Neurociencias, Universidad Miguel Hernández-CSIC, Avda. de Ramón y Cajal s/n, San Juan de Alicante, 03550 Alicante, Spain; [email protected] (F.N.); [email protected] (A.G.); [email protected] (J.M.) 4Red Temática de Investigación Cooperativa en Salud (RETICS-Trastornos Adictivos), Instituto de Salud Carlos III, MICINN and FEDER, 28029 Madrid, Spain *Correspondence: [email protected] Abstract: Alcohol binge drinking (BD) and poor nutritional habits are two frequent behaviors among many adolescents that alter gut microbiota in a pro-inflammatory direction. Dysbiotic changes in the gut microbiome are observed after alcohol and high-fat diet (HFD) consumption, even before obesity onset. In this study, we investigate the neuroinflammatory response of adolescent BD when combined with a continuous or intermittent HFD and its effects on adult ethanol consumption by using a self-administration (SA) paradigm in mice. The inflammatory biomarkers IL-6 and CX3CL1 were measured in the striatum 24 h after BD, 3 weeks later and after the ethanol (EtOH) SA. Adolescent BD increased alcohol consumption in the oral SA and caused a greater motivation to seek the substance. Likewise, mice with intermittent access to HFD exhibited higher EtOH consumption, while the opposite effect was found in mice with continuous HFD access. Biochemical analyses showed that after BD and three weeks later, striatal levels of IL-6 and CX3CL1 were increased. In addition, in saline-treated mice, CX3CL1 was increased after continuous access to HFD. After oral SA procedure, striatal IL-6 was increased only in animals exposed to BD and HFD. In addition, striatal CX3CL1 levels were increased in all BDand HFD-exposed groups. Overall, our findings show that adolescent BD and intermittent HFD increase adult alcohol intake and point to neuroinflammation as an important mechanism modulating this interaction. Keywords: binge drinking; alcohol; high-fat diet; binge; microbiota; cytokines; inflammation 1. Introduction Alcohol is the most commonly abused substance worldwide and its consumption constitutes a public health concern and a tremendous economic burden [ 1 ]. Alcohol use is commonly initiated during adolescence by engaging in intermittent episodes of binge drinking (BD) alternated with abstinence periods [ 2 ]. This pattern of intake is defined as the consumption of at least five drinks for males and four drinks for females, reaching a blood alcohol concentration of 0.08 mg/dL or above in about a 2-h period [ 3 ]. BD has been associated with poor academic performance, psychosocial problems and physical injury [ 4 , 5 ]. In addition, an early onset of BD increases the risk for developing substance use disorders later in life, particularly alcohol use disorder (AUD) [6–8]. Adolescents and young adults are especially vulnerable to the deleterious consequences of BD due to the brain maturational processes that occur during this developInt. J. Mol. Sci. 2021,22, 5279. https://doi.org/10.3390/ijms22105279 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021,22, 5279 2 of 23 mental period [ 9 , 10 ]. A large body of research has shown that BD during adolescence perturbs the structure and functioning of the prefrontal cortex and forebrain areas involved in reward [ 11 – 13 ]. These persisting alterations on brain regions are thought to underlie the long-term behavioral deficits associated with a greater vulnerability to develop an AUD [14–16]. In the last few years, the gut-brain axis has been highlighted as an important modulator of brain function and behavior [ 17 ]. Although the gut microbial community is sensitive to many factors, diet has been shown to be the primary factor shaping microbial diversity and composition [ 18 ]. Due to the ever-growing poor nutritional habits, the effects of an increased intake of food rich in both saturated fats and sugar on the microbiota is a subject of utmost concern. The consumption of a high-fat diet (HFD) is a dietary condition that changes the intestinal microbiome communities, even before the onset of obesity [ 19 ]. The gut microbiota can have indirect and direct effects in the central nervous system by acting on many pathways, primarily the regulation of inflammatory mediators [ 20 ]. Interestingly, clinical and experimental studies have reported elevated levels of inflammatory cytokines—(interleukin-6 (IL-6), interleukin-1beta (IL-1b), tumor necrosis factor alpha (TNFa)—associated with obesity or HFD consumption. Animal models of diet-induced obesity have demonstrated that consumption of HFD induces a central proinflammatory response as early as 3 days after [ 21 , 22 ]. Increased inflammatory signaling has been observed in areas in the brain such as the hypothalamus, amygdala, hippocampus and the cerebellum. Interestingly, these biomarkers of central inflammation correlated with cognitive deficits and anxiety symptomatology [23,24]. In parallel, alcohol consumption has been associated with dysbiotic changes in gut microbiome, breakdown of the intestinal barrier integrity and increased permeability of the intestinal mucosa [ 25 , 26 ]. One important source of brain damage and neurodegeneration caused by alcohol use is neuroinflammation [ 27 – 31 ]. Individuals with AUD exhibit elevated levels of several plasma cytokines such as IL-6, IL-10 and TNFa. In both clinical and preclinical studies, chronic—but also acute—exposure to alcohol is sufficient to elicit an increase in cytokine release [ 32 – 34 ]. Most importantly, blocking the alcohol-induced neuroimmune response by genetic elimination of TLR4 has prevented some long-term behavioral impairments [ 35 ]. Furthermore, the involvement of the immune response in modulating the neuropathological consequences induced by adolescent BD has also been described [36–38]. Epidemiological studies have revealed a high comorbidity between AUD and overeating pathologies such as obesity and binge-eating disorder (BED) [ 39 ]. Many obese and BED patients have difficulties restraining from consuming palatable foods (high in fat, sugar and/or salt content) and engage in compulsive behaviors similar to those observed in substance abuse disorder [ 40 , 41 ]. Compulsive overeating and binge eating are two frequent features of hedonic eating facilitated by poor impulse control. Binge eating is characterized by excessive intake of highly palatable food in a short time frame accompanied by a subjective sense of loss of control [ 42 ]. Overeating and binge eating are not only present in patients of eating disorders, but are also common in the general population, especially among adolescents [ 43 ]. In addition to behavioral commonalities, the neurobiological mechanisms modulating the reinforcing properties of drugs of abuse and palatable food are largely the same [ 44 , 45 ]. Both alcohol and HFD consumption activate the mesocorticolimbic reward circuitry consisting of dopaminergic neurons projecting from the midbrain (ventral tegmental area) to the nucleus accumbens in the striatum and the prefrontal cortex [46,47]. Animal models have been useful to study how palatable food influences dopamine signaling. It is now well characterized that intermittent access to HFD induces binge eating-like behavior in rodent models [ 48 , 49 ]. These studies have shown that food reward increases dopaminergic signaling in the striatum and that palatable food intake is specifically regulated by the nucleus accumbens [ 50 , 51 ]. Moreover, using optoinhibition techniques, it has been established that ventral striatal projections mediate binge eating Int. J. Mol. Sci. 2021,22, 5279 3 of 23 behavior [ 52 ]. Evidence suggests that bingeing on HFD produces neuroadaptations similar to those of drugs of abuse [ 47 , 53 ]. Once exposed to HFD, mice brain reward circuits remain sensitized long after a return to a standard diet [ 54 ]. In this sense, consumption of HFD during adolescence may be a risk factor for the onset and escalation of excessive alcohol consumption in adulthood. The few studies that have explored how HFD bingeing impacts ethanol consumption have yielded diverging results depending on the access schedule used [ 55 , 56 ]. Some of these studies reported increases in alcohol intake after intermittent exposure to HFD [ 57 , 58 ], while other works observed a decrease in alcohol consumption following intermittent HFD [ 59 , 60 ]. The majority of studies have not included a continuous access HFD group to distinguish the possible unique effects of HFD bingeing on ethanol intake. The purpose of this study is to further investigate the interaction between BD and two different access schedules of HFD during adolescence and its association to inflammatory markers and alcohol drinking in adulthood. Considering that both HFD and ethanol induces neuroinflammatory processes in overlapping brain circuits, we measured two inflammatory markers in the striatum when their consumption was combined. The implication of cytokine IL-6 and chemokine fractalkine (CX3CL1) gene expression in the central alcohol-related immune response made these two inflammatory mediators our choice [61–63]. In order to distinguish between the short-term and long-term effects, we evaluated the effects of BD in combination with HFD by measuring the IL-6 and CX3CL1 cytokine levels at three different time points. In this way, the study aimed to evaluate (1) the acute inflammatory effects of BD and intermittent or continuous HFD, (2) the long-term inflammatory effects of BD and intermittent or continuous HFD, and (3) the effects of BD and HFD during adolescence in increasing ethanol intake using the self-administration (SA) procedure and its association with inflammatory markers. 2. Results 2.1. Bodyweight Is Increased by Continuous but Not Intermittent Access to HFD In Experiment 1, results obtained in the statistical analyses of body weight revealed an effect of the variable Weeks [F (9,387) = 624.07; p< 0.001], as mice gained weight every week as a consequence of growth. No main effect of diet on body weight was detected, showing that bingeing did not increase body weight. From week 6 onwards, all mice lost weight due to the self-administration (SA) deprivation (Figure 1A). Int. J. Mol. Sci. 2021, 22, 5279 4 of 25 Figure 1. Body weight of mice over the procedure. Mean (± SEM) amount measured animals’ body weight. (A) No differences in body weight between the standard diet (SD) and high-fat diet binge (HFDb) groups were detected; (B) Significant differences between continuous high-fat diet (HFDc) groups and SD groups. * p < 0.05; ** p < 0.01. 2.2. Intermittent Access to HFD Induces Bingeing Behavior and Continuous Access Increases Caloric Intake An escalation in the intake of the HFD was confirmed by the ANOVA. The caloric intake during binge sessions revealed an effect on the variable Weeks [F (9,180) = 199.377; p < 0.001]. The 2 h/kcal intake in session 5 was greater than session 1 and 2 (p < 0.05) (Figure 2A). At week 6, when the SA procedure began, kcal intake was greater due to the food restriction (p < 0.001). Regarding the energy consumption of mice before the ethanol SA, the ANOVA revealed an effect of the variable Diet [F (2,18) = 56.165; p < 0.001]. The weekly energy intake was higher in the HFDc-S and HFDc-E groups compared to the rest of the groups (p < 0.001 for both groups fed on SD and both groups on HFDb) (Figure 2B). Figure 2. (A) Binge sessions. Caloric intake (kcal) of high-fat diet (HFD) in the 2-h high-fat bingeeating sessions that took place on Monday, Wednesday and Friday in the high-fat diet binge (HFDb) groups. The mean (±SEM) amount of kcal consumed in 2 h of access to high-fat food in Figure 1. Body weight of mice over the procedure. Mean ( ± SEM) amount measured animals’ body weight. ( A ) No differences in body weight between the standard diet (SD) and high-fat diet binge (HFDb) groups were detected; ( B ) Significant differences between continuous high-fat diet (HFDc) groups and SD groups. * p< 0.05; ** p< 0.01. Int. J. Mol. Sci. 2021,22, 5279 4 of 23 In Experiment 2, results obtained in the statistical analyses of body weight revealed an effect of the variable Weeks [F (9,396) = 567.389; p< 0.001], Diet [F (1,44) =7.701; p< 0.01] and the interaction Diet × Weeks [F (9,396) =1.127; p< 0.05]. Although all animals gained weight across weeks (p< 0.001), from week 3 onwards, mice in the HFDc-S and HFDc-E groups showed an increased body weight with respect to the groups fed with the standard diet (SD-S and SD-E) (Figure 1B). 2.2. Intermittent Access to HFD Induces Bingeing Behavior and Continuous Access Increases Caloric Intake An escalation in the intake of the HFD was confirmed by the ANOVA. The caloric intake during binge sessions revealed an effect on the variable Weeks [F (9,180) = 199.377; p< 0.001 ]. The 2 h/kcal intake in session 5 was greater than session 1 and 2 (p< 0.05) (Figure 2A). At week 6, when the SA procedure began, kcal intake was greater due to the food restriction (p< 0.001). Int. J. Mol. Sci. 2021, 22, 5279 4 of 25 Figure 1. Body weight of mice over the procedure. Mean (± SEM) amount measured animals’ body weight. (A) No differences in body weight between the standard diet (SD) and high-fat diet binge (HFDb) groups were detected; (B) Significant differences between continuous high-fat diet (HFDc) groups and SD groups. * p < 0.05; ** p < 0.01. 2.2. Intermittent Access to HFD Induces Bingeing Behavior and Continuous Access Increases Caloric Intake An escalation in the intake of the HFD was confirmed by the ANOVA. The caloric intake during binge sessions revealed an effect on the variable Weeks [F (9,180) = 199.377; p < 0.001]. The 2 h/kcal intake in session 5 was greater than session 1 and 2 (p < 0.05) (Figure 2A). At week 6, when the SA procedure began, kcal intake was greater due to the food restriction (p < 0.001). Regarding the energy consumption of mice before the ethanol SA, the ANOVA revealed an effect of the variable Diet [F (2,18) = 56.165; p < 0.001]. The weekly energy intake was higher in the HFDc-S and HFDc-E groups compared to the rest of the groups (p < 0.001 for both groups fed on SD and both groups on HFDb) (Figure 2B). Figure 2. (A) Binge sessions. Caloric intake (kcal) of high-fat diet (HFD) in the 2-h high-fat bingeeating sessions that took place on Monday, Wednesday and Friday in the high-fat diet binge (HFDb) groups. The mean (±SEM) amount of kcal consumed in 2 h of access to high-fat food in Figure 2. (A) Binge sessions. Caloric intake (kcal) of high-fat diet (HFD) in the 2-h high-fat binge-eating sessions that took place on Monday, Wednesday and Friday in the high-fat diet binge (HFDb) groups. The mean ( ± SEM) amount of kcal consumed in 2 h of access to high-fat food in every binge session * p< 0.05; *** p< 0.001 significant difference with respect to the first and second binge sessions. ( B ) Weekly energy consumption per cage (four mice) across the procedure. Mean ( ± SEM) 24 h energy consumption (kcal) of mice (standard chow shown by open bars and HFD by solid bars) *** p< 0.001 significant difference of both continuous high-fat diet (HFDc) groups with respect to the standard diet (SD) groups and HFDb groups. Regarding the energy consumption of mice before the ethanol SA, the ANOVA revealed an effect of the variable Diet [F (2,18) = 56.165; p< 0.001]. The weekly energy intake was higher in the HFDc-S and HFDc-E groups compared to the rest of the groups (p< 0.001 for both groups fed on SD and both groups on HFDb) (Figure 2B). 2.3. A High Fat Diet and Intermittent Ethanol Intake during Adolescence Increases the Neuroinflammatory Response After the end of the ethanol BD, the ANOVA of the striatal levels of IL-6 (Figure 3A) showed an effect of the variable Diet [F (2,57) = 7.680; p= 0.001], Treatment [F (1,57) = 31.752; p= 0.001], and the interaction Diet × Treatment [F (2,57) = 3.161; p= 0.05]. Intermittent and repeated ethanol administration during adolescence increased the striatal levels of IL-6 in mice fed with HFD (either in binge or continuously) with respect to those non-exposed to ethanol (p< 0.001 in both cases). Equally, HFDc-E and HFDb-E groups presented higher striatal levels of IL-6 than mice exposed to ethanol during adolescence but fed with the standard diet (SD-E) (p< 0.001 in both cases). Int. J. Mol. Sci. 2021,22, 5279 5 of 23 Int. J. Mol. Sci. 2021, 22, 5279 6 of 25 Figure 3. Intermittent ethanol intake during adolescence and high-fat diet (HFD) increase IL-6 levels in the striatum. The columns represent the mean and the vertical lines ± SEM of concentration levels of IL-6 (pg/mg protein) (A) after ethanol binge drinking (BD) during adolescence, (B) 3 weeks after the last ethanol injection and (C) after the end of the oral self-administration (SA) procedure in the groups with food deprivation and (D) in the groups without food deprivation *** p < 0.001, ** p < 0.01 with respect to the corresponding non-ethanol-treated groups; +++ p < 0.001 with respect to the corresponding standard diet fed group. The ANOVA for the striatal levels of CX3CL1 after ethanol BD (Figure 4A) showed an effect of the variable Treatment [F (1,80) = 42.977; p = 0.001] and the interactions of Diet × Treatment [F (2,80) = 6.414; p = 0.003]. Mice fed on HFD continuously (HFDc-S) presented higher striatal level of CX3CL1 than those fed on the standard diet (p < 0.01) or intermittent HFD (p < 0.05). Ethanol exposure during adolescence increased CX3CL1 in all groups (p < 0.001 in all cases). Figure 3. Intermittent ethanol intake during adolescence and high-fat diet (HFD) increase IL-6 levels in the striatum. The columns represent the mean and the vertical lines ± SEM of concentration levels of IL-6 (pg/mg protein) ( A ) after ethanol binge drinking (BD) during adolescence, ( B ) 3 weeks after the last ethanol injection and ( C ) after the end of the oral self-administration (SA) procedure in the groups with food deprivation and ( D ) in the groups without food deprivation *** p< 0.001, ** p< 0.01 with respect to the corresponding non-ethanol-treated groups; +++ p< 0.001 with respect to the corresponding standard diet fed group. Three weeks after ethanol binge drinking, the ANOVA of the striatal levels of IL-6 (Figure 3B) showed an effect of the variable Treatment [F (1,51) = 9.332; p= 0.004]. All mice exposed to ethanol during adolescence showed higher levels of IL-6 (p< 0.01). In groups exposed to food deprivation during ethanol SA, the ANOVA of the striatal levels of IL-6 (Figure 3C) showed an effect of the variable Diet [F (1,33) = 4.308; p= 0.046], Treatment [F (1,33) = 7.128; p= 0.012], and the interaction Diet × Treatment [F (1,33) = 4.278; p= 0.047]. Mice exposed to ethanol during adolescence and fed on HFD intermittently (HFDb-E) showed higher striatal levels of IL-6 after ethanol SA than the rest of the groups (p< 0.01 in all cases). In groups that performed ethanol SA without food deprivation, the ANOVA of the striatal levels of IL-6 (Figure 3D) showed an effect of the variable Treatment [ F (1,47) = 7.438 ; Int. J. Mol. Sci. 2021,22, 5279 6 of 23 p= 0.009]. Groups exposed to ethanol during adolescence showed higher IL-6 levels (p< 0.01), although the effect was mainly due to the group fed on continuous HFD. The ANOVA for the striatal levels of CX3CL1 after ethanol BD (Figure 4A) showed an effect of the variable Treatment [F (1,80) = 42.977; p= 0.001] and the interactions of Diet ×Treatment [F (2,80) = 6.414; p= 0.003]. Mice fed on HFD continuously (HFDc-S) presented higher striatal level of CX3CL1 than those fed on the standard diet (p< 0.01) or intermittent HFD (p< 0.05). Ethanol exposure during adolescence increased CX3CL1 in all groups (p< 0.001 in all cases). Int. J. Mol. Sci. 2021, 22, 5279 7 of 25 Figure 4. Intermittent ethanol intake during adolescence and continuous high-fat diet (HFD) increase CX3CL1 levels in the striatum. The columns represent the mean and the vertical lines ± SEM of concentration levels of CX3CL1 (ng/mg protein) (A) after ethanol BD during adolescence, (B) 3 weeks after the last ethanol injection and (C) after the end of the oral self-administration (SA) procedure in the groups with food deprivation and (D) in the groups without food deprivation *** p < 0.001, * p < 0.05 with respect to the corresponding non-ethanol-treated groups; +++ p < 0.001, ++ p < 0.01 with respect to the corresponding standard diet fed group. # p < 0.05 with respect to the corresponding binge HFD group. Similar results revealed the ANOVA for the striatal levels of CX3CL1 three weeks after the ethanol BD with an effect of the variable Treatment [F (1,54) = 39.365; p = 0.001], Diet [F (2,54) = 3.627; p = 0.033], and the interactions of Diet × Treatment [F (2,54) = 3.799; p = 0.029], (Figure 4B). Mice fed on HFD continuously (HFDc-S) presented higher striatal level of CX3CL1 than those fed on the standard diet or HFDb-S (p < 0.01 in both cases). Ethanol exposure during adolescence increased CX3CL1 in all groups (p < 0.001 in all cases). In groups exposed to food deprivation during ethanol SA, the ANOVA of the striatal levels of CX3CL1 (Figure 4C) showed an effect of the variable Treatment [F (1,35) = 41.498; p = 0.001], and the interaction Diet × Treatment [F (1,35) = 9.504; p = 0.004]. Mice exposed to ethanol during adolescence showed higher striatal levels of CX3CL1 after ethanol SA than those treated with saline (p < 0.001 for SD-S and p < 0.05 for HFDb-S). However, those intermittently fed with HFD (HFDb-S) showed higher CX3CL1 levels than those fed only with the standard diet (p < 0.01). Figure 4. Intermittent ethanol intake during adolescence and continuous high-fat diet (HFD) increase CX3CL1 levels in the striatum. The columns represent the mean and the vertical lines ± SEM of concentration levels of CX3CL1 (ng/mg protein) ( A ) after ethanol BD during adolescence, ( B ) 3 weeks after the last ethanol injection and ( C ) after the end of the oral self-administration (SA) procedure in the groups with food deprivation and ( D ) in the groups without food deprivation *** p< 0.001 , *p< 0.05 with respect to the corresponding non-ethanol-treated groups; +++ p< 0.001, ++ p< 0.01 with respect to the corresponding standard diet fed group. # p< 0.05 with respect to the corresponding binge HFD group. Similar results revealed the ANOVA for the striatal levels of CX3CL1 three weeks after the ethanol BD with an effect of the variable Treatment [F (1,54) = 39.365; p= 0.001], Diet [F (2,54) = 3.627; p= 0.033], and the interactions of Diet × Treatment [F (2,54) = 3.799; p= 0.029 ], (Figure 4B). Mice fed on HFD continuously (HFDc-S) presented higher striatal Int. J. Mol. Sci. 2021,22, 5279 7 of 23 level of CX3CL1 than those fed on the standard diet or HFDb-S (p< 0.01 in both cases). Ethanol exposure during adolescence increased CX3CL1 in all groups (p< 0.001 in all cases). In groups exposed to food deprivation during ethanol SA, the ANOVA of the striatal levels of CX3CL1 (Figure 4C) showed an effect of the variable Treatment [F (1,35) = 41.498; p= 0.001], and the interaction Diet × Treatment [F (1,35) = 9.504; p= 0.004]. Mice exposed to ethanol during adolescence showed higher striatal levels of CX3CL1 after ethanol SA than those treated with saline (p< 0.001 for SD-S and p< 0.05 for HFDb-S). However, those intermittently fed with HFD (HFDb-S) showed higher CX3CL1 levels than those fed only with the standard diet (p< 0.01). In groups that performed ethanol SA without food deprivation the ANOVA of the striatal levels of CX3CL1 showed an effect of the variable Treatment [F (1,53) =23.123; p= 0.001 ], and the interaction Diet × Treatment [F (1,53) =9.421; p= 0.003] (Figure 4D). Ethanol exposure during adolescence induced higher CX3CL1 levels (p< 0.001 in all cases). The group fed on continuous HFD and treated with saline also showed increased levels of CX3CL1 with respect to SD-S (p< 0.001). 2.4. Oral Self-Administration of Ethanol 2.4.1. Experiment 1 The ANOVA for the EtOH consumption (g/kg) during the FR1 schedule revealed a significant effect of the variable Treatment [F (1,46) = 9.860; p< 0.01] and the interaction Days × Diet [F (4,184) = 2.503; p< 0.05] (Figure 5A). Animals that received BD treatment during adolescence (SD-E and HFDb-E) exhibited increased alcohol consumption compared to those that received saline injections (SD-S and HFD-S) (p< 0.01). Also, mice in the HFDb groups (both HFDb-E and HFDb-S) exhibited an increased ethanol oral SA of EtOH (6%) in day 1 compared to day 2 (p< 0.01), day 3 (p< 0.05) and day 4 (p< 0.01). When both saline groups (SD-S and HFDb-S) were independently analyzed in order to confirm the effect of HFD in ethanol intake, the ANOVA revealed a significant effect of the variable Diet [F (1,24) = 6.065 ;p< 0.05]. Mice in the HFDb-S group exhibited increased ethanol consumption compared to the SD-S group (p< 0.05). With respect to the number of effective responses during FR1, the ANOVA revealed a significant effect of the variable Treatment [F (1,47) = 5.950; p< 0.05], (Figure 5B). Mice who received ethanol treatment (SD-E and HFDb-E) performed more effective responses compared to those who received saline (p< 0.05). During the FR3 schedule, the ANOVA for the ethanol consumption revealed a significant effect of the interaction Days × Diet × Treatment [F (4,184) = 6.953 p< 0.001] (Figure 5A) . Post-hoc comparisons showed that animals in the HFDb-E group presented increased ethanol intake compared to the HFDb-S group on days 7 (p< 0.05), 8 (p< 0.001), 9 and 10 (p< 0.5 for both). Moreover, animals in the SD-E group exhibited an increased alcohol consumption compared to the SD-S group on days 9 and 10 (p< 0.05 for both). With respect to effective responses, the ANOVA revealed a significant effect of the interaction Days × Diet × Treatment [F (4,184) =4.223; p< 0.01] (Figure 5B). Mice in the SD-E group performed more effective responses than the SD-S on days 9 and 10 (p< 0.05 for both). Similarly, animals in the HFDb-E group performed more effective responses than the HFD-S group on days 7, 8 (p< 0.5) and 10 (p< 0.001). Analyses of the PR showed an effect of the variable Treatment [F (1,42) = 16.456; p< 0.001 ] in the motivation to seek alcohol (Figure 5C). Animals that received BD treatment during adolescence (SD-E and HFDb-E) presented higher breaking point values compared to the saline groups (p< 0.001). In reference to ethanol consumption, results showed an effect of the interaction Diet × Treatment [F (1,42) = 4.194; p< 0.05], (Figure 5D). Animals on the SD-E group exhibited an increased ethanol consumption than animals in the SD-S group (p< 0.05). Int. J. Mol. Sci. 2021,22, 5279 8 of 23 Int. J. Mol. Sci. 2021, 22, 5279 9 of 25 Figure 5. Effects of intermittent high-fat diet (HFD) bingeing on effective responses and oral ethanol EtOH self-administration in OF1 mice. Data are presented as mean (± SEM): (A) g/kg of 6% EtOH consumption and (B) the number of effective responses during FR1 and FR3. The columns represent means and the vertical lines ± SEM of (C) breaking point values during PR and (D) amount of 6% EtOH consumption during the PR session. * p < 0.05; ** p < 0.01; *** p < 0.001 values that are significantly different from animals that received BD compared to the saline groups. + p < 0.05; +++ p < 0.001 significant differences in the HFDb-E compared to the HFD-S group. @ p < 0.05 significant differences in SD-E group with respect to SD-S. # p < 0.05 significant differences in the HFDb group between Day 1 compared to Day 2, 3 and 4. During the FR3 schedule, the ANOVA for the ethanol consumption revealed a significant effect of the interaction Days × Diet × Treatment [F (4,184) = 6.953 p < 0.001] (Figure 5A). Post-hoc comparisons showed that animals in the HFDb-E group presented increased ethanol intake compared to the HFDb-S group on days 7 (p < 0.05), 8 (p < 0.001), 9 and 10 (p < 0.5 for both). Moreover, animals in the SD-E group exhibited an increased alcohol consumption compared to the SD-S group on days 9 and 10 (p < 0.05 for both). With respect to effective responses, the ANOVA revealed a significant effect of the interaction Days × Diet × Treatment [F (4,184) =4.223; p < 0.01] (Figure 5B). Mice in the SD-E group Figure 5. Effects of intermittent high-fat diet (HFD) bingeing on effective responses and oral ethanol EtOH selfadministration in OF1 mice. Data are presented as mean ( ± SEM): ( A ) g/kg of 6% EtOH consumption and ( B ) the number of effective responses during FR1 and FR3. The columns represent means and the vertical lines ± SEM of ( C ) breaking point values during PR and ( D ) amount of 6% EtOH consumption during the PR session. * p< 0.05; ** p< 0.01; *** p< 0.001 values that are significantly different from animals that received BD compared to the saline groups. + p< 0.05; +++ p< 0.001 significant differences in the HFDb-E compared to the HFD-S group. @ p< 0.05 significant differences in SD-E group with respect to SD-S. # p< 0.05 significant differences in the HFDb group between Day 1 compared to Day 2, 3 and 4. 2.4.2. Experiment 2 For EtOH consumption (g/kg), the ANOVA of the FR1 schedule revealed a significant effect of the interaction Days × Diet × Treatment [F (4,164) = 2.658 p< 0.05], (Figure 6A) . On day 1, mice in the SD-E group exhibited an increased ethanol oral SA of EtOH (6%) with respect to the SD-S group (p< 0.05). With respect to the number of effective responses during FR1, the ANOVA revealed a significant effect of the variable Treatment [ F (1,41) = 7.136 ;p< 0.05] (Figure 6B). Animals that received BD treatment during adoles- Int. J. Mol. Sci. 2021,22, 5279 9 of 23 cence (SD-E and HFDc-E) presented increased effective responses with respect to the saline groups (p< 0.05). Int. J. Mol. Sci. 2021, 22, 5279 11 of 25 Figure 6. Effects of continuous access to high-fat diet (HFD) on effective responses and oral ethanol EtOH self-administration in OF1 mice. Data are presented as mean (± SEM): (A) g/kg of 6% EtOH consumption (B) the number of effective responses during FR1 and FR3. The columns represent means and the vertical lines ± SEM of (C) breaking point values during PR and (D) amount of 6% EtOH consumption during the PR session * p < 0.05 significant differences in the groups that received BD (SD-E and HFD-E) compared to the saline groups (SD-S and HFDc-S).+ p < 0.05 significant differences between SD-E with respect to SD-S. # p < 0.05 significant differences between the BD-treated groups and the saline-treated groups in Day 9. ## p < 0.01 significant differences between the HFDc groups (HFDc-S and HFDc-E) compared to the SD groups (SD-S and SD-E). The ANOVA for the ethanol consumption during FR3 schedule revealed a significant effect of the interaction Days × Diet × Treatment [F (4,156) = 3.853 p < 0.01] (Figure 6A). Mice in the SD-E group showed an increased ethanol consumption rate compared to those in the SD-S group on days 9 and 10 (p < 0.05 for both). With respect to effective responses, the ANOVA revealed a significant effect of the interaction Days × Treatment [F (4,172) = 3.126; p < 0.05] (Figure 6B). Animals that received BD treatment presented more effective responses with respect to the saline groups on day 9 (p < 0.01). Analyses of the PR revealed no differences in the motivation to seek alcohol between groups (Figure 6C). In reference to ethanol consumption, results showed an effect of the Figure 6. Effects of continuous access to high-fat diet (HFD) on effective responses and oral ethanol EtOH self-administration in OF1 mice. Data are presented as mean ( ± SEM): ( A ) g/kg of 6% EtOH consumption ( B ) the number of effective responses during FR1 and FR3. The columns represent means and the vertical lines ± SEM of ( C ) breaking point values during PR and ( D ) amount of 6% EtOH consumption during the PR session * p< 0.05 significant differences in the groups that received BD (SD-E and HFD-E) compared to the saline groups (SD-S and HFDc-S). + p< 0.05 significant differences between SD-E with respect to SD-S. # p< 0.05 significant differences between the BD-treated groups and the saline-treated groups in Day 9. ## p< 0.01 significant differences between the HFDc groups (HFDc-S and HFDc-E) compared to the SD groups (SD-S and SD-E). The ANOVA for the ethanol consumption during FR3 schedule revealed a significant effect of the interaction Days × Diet × Treatment [F (4,156) = 3.853 p< 0.01] (Figure 6A ). Mice in the SD-E group showed an increased ethanol consumption rate compared to those in the SD-S group on days 9 and 10 (p< 0.05 for both). With respect to effective responses, the ANOVA revealed a significant effect of the interaction Days × Treatment Int. J. Mol. Sci. 2021,22, 5279 16 of 23 the weight at 80% of the pre-training body weight. In Experiment 2, food and water were available ad libitum throughout the procedure. 4.5.2. Saccharin Fading (9 Days) The saccharin concentration was gradually decreased as the EtOH concentration was gradually increased [ 133 , 134 ]. Each solution combination was set up to three consecutive sessions per combination (0.15% Sac − 2% EtOH; 0.10% Sac − 4% EtOH; 0.05% Sac −6% EtOH). 4.5.3. Ethanol Consumption 6% (11 Days) The aim of the last phase was to evaluate the number of responses on the active nose-poke and the 6% EtOH (w/v) consumption. During 5 daily consecutive test sessions the number of active responses and EtOH consumption ( µ L) was measured under a fixed-ratio (FR)-1 followed by another five consecutive days under a FR3-schedule (three responses on the active nose-poke were needed to obtain one reinforcement). Alcohol intake was determined by subtracting the alcohol leftover in the receptacle collected with a micropipette after each session. 4.5.4. Progressive Responding Ratio for Alcohol In the last session we tested the motivation to seek alcohol by setting a progressive ratio (PR) paradigm. In the PR-schedule the response requirement to obtain a reinforcement escalated according to the following series: 1-2-3-5-12-18-27-40-60-90-135-200-300-450-6751000. The breaking point for each animal was defined as the highest number of nose-pokes each animal performed to earn one reinforcement during the 2 h session. 4.6. Tissue Sampling To obtain tissue samples, mice were sacrificed by cervical dislocation 24 h after the ethanol consumption and 48 h after the binge session. Brains were rapidly removed, and the striatum was dissected out in both hemispheres following the procedure described by Heffner et al. [135], after which it was flash frozen in dry ice until storage at −80 ◦C. 4.7. IL-6 and CX3CL1 Measurements Frozen brain striatal nuclei were homogenized in 250 mg of tissue/0.5 mL of cold lysis buffer (1% NP-40, 20 mM Tris-HCl pH 8, 130 mM NaCl, 10 mM NaF, 10 µ g/mL aprotinin, 10 µ g/mL leupeptin, 40 mM DTT, 1 mM Na3VO4, and 10 mM PMSF). Brain homogenates were kept on ice for 30 mim. and centrifuged at maximum speed for 15 min, after being determined by the Bradford assay from ThermoFisher (Waltham, MA, USA). Striatal IL-6 and CX3CL1 concentrations were quantified by using an enzyme-linked immunosorbent essay (Mouse IL-6 ELISA Kit, ab 100712; Mouse Fractalkine ELISA Kit, ab100683) following the manufacturer’s protocol (Abcam, Cambridge, UK). To determine absorbance, we employed an iMark microplate reader (Bio-RAD, Hercules, CA, USA) controlled by Microplate Manager 6.2 software. The optical density was read at 450 nM and the final results were calculated using a standard curve carried according to the manufacturer’s instructions. The data were expressed as pg/mL for plasma, and pg/mg for tissue samples. 4.8. Statistical Analyses In both experiments, data relating to body weight was analyzed by a two-way ANOVA with a two between-subject’s variable -Treatment (saline and ethanol) and Diet (standard diet (SD) and continuous high-fat diet (HFDc) or binge high-fat diet (HFDb))- and a within variable Weeks with 10 levels (from PND 25 to 88). The total weekly energy intake was analyzed by a two-way ANOVA with two between variables -Treatment (saline and ethanol) and Diet (standard diet (SD) and continuous high-fat diet (HFDc) or binge high-fat diet (HFDb)). Intake during bingeing sessions was analyzed by a one-way ANOVA with one Int. J. Mol. Sci. 2021,22, 5279 17 of 23 between-subject’s variable—Treatment (saline and ethanol) and a within variable Weeks with 10 levels. In Experiment 1, EtOH SA and effective responses were analyzed individually by a two-way ANOVA with a two between-subject’s variable -Treatment (saline and ethanol) and Diet (standard diet (SD), high-fat binge (HFDb)- and a within-subject’s variable—Days, with five levels of FR1 or FR3 schedule. In addition, differences between the saline groups in the FR1 were tested with a two-way ANOVA with one between-subject’s variable Diet (standard diet (SD), high-fat binge (HFDb)- and a within-subject’s variable—Days, with five levels of FR1. A two-way ANOVA with a two between-subject’s variable—Treatment (saline and ethanol) and Diet (standard diet (SD), high-fat binge (HFDb)) was employed to analyze ethanol consumption and breaking point values during the PR session. In Experiment 2, two two-way ANOVAs were performed with a two between-subject’s variable—Treatment (saline and ethanol) and Diet (standard diet (SD), continuous high-fat diet (HFDc)- and a within-subject’s variable -Days, with five levels of FR1 or FR3 schedule. A two-way ANOVA with a two between-subject’s variable—Treatment (saline and ethanol) and Diet (standard diet (SD), continuous high-fat diet (HFDc)) was employed to analyze ethanol consumption and breaking point values during the PR session. A two-way ANOVA was employed to analyze the data from the biochemical parameters (ELISA assays for the CX3CL1 and IL-6) with a two between-subject’s variable, previously described, Treatment and Diet. For the data obtained after the self-administration procedure, two different ANOVAs were performed for Experiment 1 and 2. In all cases, post-hoc comparisons were performed with Bonferroni tests. All statistical analyses were performed using SPSS Statistics v26. Data were expressed as mean ± SEM and a value of p< 0.05 was considered statistically significant. Author Contributions: Conceptualization, M.R.-A., S.M.-R. and J.M. (JoséMiñarro); methodology M.G.-P., F.N. and A.G.; software M.G.-P. and S.M.-R.; validation M.R.-A. and J.M. (JoséMiñarro); formal analysis S.M.-R. and M.R.-A.; investigation M.G.-P., F.N., A.G. and S.M.-R.; resources M.R.-A., J.M. (JoséMiñarro) and J.M. (Jorge Manzanares); data curation M.R.-A., M.G.-P. and S.M.-R.; writing— original draft preparation, S.M.-R. and M.G.-P.; writing—review and editing, M.G.-P., M.R.-A., J.M. (JoséMiñarro), J.M. (Jorge Manzanares); visualization M.G.-P.; supervision M.R.-A. and S.M.-R.; project administration M.R.-A., J.M. (JoséMiñarro) and J.M. (Jorge Manzanares).; funding acquisition M.R.-A., J.M. (JoséMiñarro) and J.M. (Jorge Manzanares). All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Spanish Ministry of Health, Social Affairs and Equality, Government Delegation for the National Drugs Plan (2018/013 to MRA and PNSD 2016I016 to JMa); Generalitat Valenciana, Conselleria de Educación, Dirección General de Universidades, Grupos de Investigación de excelencia, PROMETEOII/2018/132 to JMi; Ministerio de Economía y Competitividad’ (FIS, PI14/00438) to JMa. Instituto de Salud Carlos III, Red de Trastornos Adictivos (RD16/0017/0007 to JMi) and Unión Europea, Fondos FEDER “una manera de hacer Europa”. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of the University of Valencia (2017/VSC/PEA/00204). 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