Mediterranean X Western based diets: Opposite influences on opioid reinstatement

Toxicology Letters 308 (2019) 7–16

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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

Mediterranean X Western based diets: Opposite influences on opioid reinstatement

T

Laura Hautrive Milanesia, Domenika Rubert Rossatoc, Verônica Tironi Diasa, Maikel Kronbauera, Lívia Ferraz D’avilaa, Sabrina Somacalb, Thiago Duartea, Marta Maria Frescura Duartea,d, ⁎ Tatiana Emanuellia,c, Marilise E. Burgera,c, a

Programa de Pós-Graduação em Farmacologia, Universidade Federal de Santa Maria (UFSM), RS, Brazil Programa de Pós-Graduação em Ciência e Tecnologia dos Alimentos, Universidade Federal de Santa Maria (UFSM), RS, Brazil c Departamento de Fisiologia e Farmacologia, UFSM, RS, Brazil d Universidade Luterana do Brasil (ULBRA), Santa Maria, RS, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Opioid addiction Conditioned place preference Diets Interesterified fat Palm oil HPA axis

Opioids are addictive drugs, whose misuse evoke withdrawal and relapse. Mediterranean-based diet (MBD) is rich in n-3 polyunsaturated fatty acids (PUFA), while Western based diets (WBDs) contain saturated fatty acids including interesterified fat (IF) and palm oil (PO), influencing neural functions. We compared MBD and WBDs on morphine-induced addiction parameters. Rats fed with MBD (chow plus 20% soybean- and fish-oil- n-6/n-3 PUFA 1:1) or WBD (WBD- PO or WBD-IF: chow plus 20% of palm oil or interesterified fat, respectively; high n-6/ n-3 PUFA ratio) were exposed to morphine in conditioned place preference (CPP) paradigm. Anxiety-like behavior, locomotion and thermal sensitivity were evaluated during withdrawal. After morphine-CPP extinction, animals were challenged to morphine-reinstatement to induce relapse. All groups showed morphine-CPP, WBDs favored anxiety-like behaviors per se, locomotor sensitization and thermal hipersensitivity during withdrawal, resulting in increased morphine-reinstatement in comparison to MBD, which did not show relapse. WBDs increased glucocorticoid receptor immunoreactivity in the pre-frontal cortex, increasing corticosterone (CORT) and adrenocorticotrophic hormone (ACTH) per se and after morphine-reinstatement. In the nucleus accumbens, WBDs increased dopamine transporter (DAT) and dopamine receptor-2 (D2R) immunoreactivity and decreased dopamine receptor-1 (D1R). These findings indicate that WBDs facilitate morphine-reinstatement, unlike MBD, preserving the DA system mesolimbic neuroplasticity.

1. Introduction Opioid addiction is a chronic relapsing disorder characterized by the uncontrolled drive to obtain the substance and failure to limit its use despite adverse, often severe consequences (Kalivas et al., 2005). In fact, opioids have highly addictive power and a broad range of neuroadaptations developed in response to chronic exposure, which are thought to be critical for the expression of opioid addiction main characteristics: tolerance, anxiety, thermal hypersensitivity, drug seeking and processes that may contribute to reinstatement (Milton and Everitt, 2012; Koob and Le Moal, 2005). Morphine, a main opioid representative, stimulates DA transmission and causes changes in the mesocorticolimbic brain areas, which play a critical role in regulating drug-seeking behaviors (Reisi et al., 2014). The relapse is the major

challenge for the treatment of opioid addiction and can be elicited by the exposure to the drug itself, by drug associated cues or by behavioral changes such as anxiety and restlessness during drug withdrawal (Bocarsly et al., 2011). The neural circuitry underlying reward, emotion and addictive processes includes the mesolimbic pathway and dopaminergic system, which may be estimated by dopamine receptors (D1R-D5R) and transporter (DAT), particularly in the nucleus accumbens (NAc) (Geiger et al., 2009). The brain's reward pathway is closely involved in drug addiction and can be modified through eating habits, particularly those with high fat diets (Carter et al., 2016). High fat diets are rewarding and their intake can recruit neural pathways and mechanisms linked to addiction (Sharma et al., 2013). Fatty acids are essential components present in the phospholipid

⁎ Corresponding author at: Graduation Program of Pharmacology, Physiology and Pharmacology Department, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, 97105-900, Brazil. E-mail address: [email protected] (M.E. Burger).

https://doi.org/10.1016/j.toxlet.2019.03.009 Received 6 December 2018; Received in revised form 11 February 2019; Accepted 17 March 2019 Available online 18 March 2019 0378-4274/ © 2019 Elsevier B.V. All rights reserved.

Toxicology Letters 308 (2019) 7–16

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Fig. 1. Experimental design: Three experimental groups of rats were fed from weaning (PND 21) until the end of experiment (PND 92) with diets enriched with different types of fatty acids. The animals were conditioned with morphine (4 mg/kg, i.p.) in CPP protocol and assessed in extinction test in CPP apparatus, anxiety-like symptoms (EPM), locomotor activity (OF) and thermal sensitivity (HP) related to drug withdrawal. After morphine-CPP extinction the animals were challenged in morphine-reinstatement (2 mg/kg, i.p.) in CPP apparatus. Abbreviation: MBD: Mediterranean-based diet; WBD: Western based diet; PO: palm oil; IF: interesterified fat; PND: postnatal day; CPP: conditioned place preference; EPM: elevated plus maze; OF: open field; HP: hot plate; Ext: extinction.

2. Materials and methods

cell membranes structure and they are abundant in the central nervous system (CNS) (Cotman et al., 1969). In fact, essential fatty acids (AGE) are polyunsaturated fatty acids (PUFA) and are recognized as essential due to the inability of mammals to synthesize them, being only obtained through diet (Zararsiz et al., 2006). The Mediterranean-based diet (MBD) is recognized by its benefits due to its balanced level of PUFA, presenting an n-6/n-3 PUFA ratio of about 1:1 (Simopoulos, 2002; Morris et al., 2015). Throughout evolution, the consumption of EFA, rich in n-3 PUFA has decreased in Western Countries where an n6/n-3 PUFA ratio is currently around 15:1 (Simopoulos, 2002), while the acceptable limits are about 10:1 (Institute of Medicine-DRIs, 2002). The current Western diet is characterized by a large quantity of processed foods, rich in trans fatty acids, saturated fatty acids (SFA) and n-6 PUFA (Kanoski and Davidson, 2011). SFA-enriched diet can have deleterious metabolic actions that include increasing visceral fat mass, type II diabetes (Rosqvist et al., 2014) and central deleterious effects such as increased anxiety (de Wit et al., 2010). As observed in Western countries, a nutritional n-3 PUFA deficiency favors an imbalance of the n-6/n-3 PUFA ratio, which is a risk factor for CNS diseases development (Pase et al., 2015), HPA axis activation, neuropsychiatric diseases including anxiety (Larrieu et al., 2016) and drug addiction (Kuhn et al., 2015a, b); however, its pathophysiological mechanisms remain poorly understood. Over the years, our research group has demonstrated through several studies that the trans fat from hydrogenated vegetable fat can be incorporated into neural membranes, increasing the risk of developing neuropsychiatric conditions (Trevizol et al., 2014; Pase et al., 2013, 2015) and modifying addiction parameters (Kuhn et al., 2015a, b; Roversi et al., 2016). In view of several deleterious health effects related to trans fat consumption, this fat has been industrially replaced by the interesterified fat (IF) and palm oil (PO) (Basu et al., 2013; Sen et al., 2007). In this context, palm oil (PO), which is mainly extracted from the African palm fruit, (Elaeis guineensis Jacq.) has high SFA content, specifically palmitic acid (C16:0, ∼42.3%), as well as the IF (Kien et al., 2013), which besides SFA, is also rich in interesterified FA. Considering the current diets containing interesterified FA, few studies about the consequences of its chronic consumption on the CNS functions have been developed (D’Avila et al., 2017; Milanesi et al., 2017; Haygert et al., 2018). It has been proposed that dopaminergic neurotransmission can be modified by the consumption of palatable foods (Norgren et al., 2006) and can result in associations between environmental cues with reward effects (Volkow et al., 2011). Considering the current Western dietary habits, we compared the influence of western-based diets (WBD), which were enriched with Palm oil (WBD-PO) or interesterified fat (WBD-IF), rich in SFA and interesterified FA, respectively, in relation to the Mediterranean-based diet (with ideal n-6/n-3 PUFA) on the morphine addictive properties as well as on drug relapse factors, including behaviors and molecular adaptations involving the plasticity of the dopaminergic mesolimbic system and HPA axis.

2.1. Animals Forty-two male Wistar rats (21 day-old) from the breeding facility of the Federal University of Santa Maria (UFSM), RS, Brazil, were kept in Plexiglas cages with free access to food and water in a room with controlled temperature (22 ± 2 °C) and on a 12 h-light/dark cycle. This study was approved by the Animal Ethics Committee of the Federal University of Santa Maria (7948130517-UFSM), affiliated to the Council for the Control of Animal Experiments (CONCEA). 2.2. General procedures Animals were randomly assigned to one of the three experimental groups (n = 14 for each group). They received enriched chow with 20% additional fat or oil (Teixeira et al., 2012; Pase et al., 2015), from the postnatal day 21 (PND21) until the end of the experiment (PND 92) resulting in 10 weeks of diet. The different fats and oil resulted in three personalized diets: a Mediterranean-based diet (MBD- n-6/n-3 PUFA ratio 1:1), Western based diet containing PO (WBD1-PO 20%) and Western based diet containing Interesterified Fat (WBD2-IF 20%). The animals’ body weight was weekly monitored. After 8 weeks, the animals on diet were submitted to behavioral tests and subdivided in six experimental groups (n = 7): MBD-Vehicle; MBD-Morphine; WBD-POVehicle; WBD-PO-Morphine; WBD-IF-Vehicle; WBD-IF-Morphine (Fig. 1). Following (48 h) the last behavioral assessments, all animals were anesthetized (isofluorane) and euthanized by exsanguination. The ratio of adrenal weight (AW/BW) and epididymal fat weight (EFW/BW) to body weight was measured after the end of the experiment. For removal of the adrenal glands and epididymal fat, the carcasses were placed on a dissecting board on dorsal recumbency after each incision was made from the linea alba to the anal region thereby exposing the visceral organs. The adrenal glands are located in the posterior abdomen, over the medial aspect of the upper poles of each kidney (Olukole et al., 2016). The adrenal glands were removed and weighed. In the same incision, the epididymal fat was dissected. This fat is located around the epididymis, which is a conjoined tubule located above each testis. Each fatty deposit of the epididymis was dissected and immediately weighed (ChusWang et al., 2016). 2.3. Diet composition The diets were isocaloric and normolipid. The only difference among them was the FA quality. All three diets consisted of standard chow (PUROLAB 22®, Puro Trato Nutrição Animal Ltda) enriched with 20% additional fat or oil: i) soybean oil (SO, rich in n-6 FA), (Camera®, Ijuí, Brazil), purchased from a local supermarket plus fish oil (FO, rich in n-3 FA), donated by Laboratório Tiaraju® (Santo Ângelo, Brazil); ii) PO (rich in SFA Palmitic acid C16:0), (Agropalma®, São Paulo, Brazil); 8

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vehicle in the paired compartment, with an interval of 6 h between each conditioning. The control group was injected with the vehicle (0.9% NaCl, i.p.) in both sides of the apparatus. On the testing day, rats were individually placed in the center of the chosen chamber with free access to both compartments for 15 min, without any injection. Time spent in the drug-paired environment was interpreted as morphine preference. For morphine-CPP extinction, the animals were placed in the CPP apparatus every other day and were allowed to freely access all compartments for 15 min. The time spent in each compartment was recorded and the conditioning score was calculated in the same way as in the pre-test and test. This procedure was repeated until the calculated conditioning scores in two consecutive extinction tests became similar to those on the pre-conditioning day. After morphine-CPP extinction animals were challenged to morphine-reinstatement to induce drug relapse. The reinstatement test was performed on the day following a successful extinction test (Li et al., 2013). On the reinstatement day, animals received the vehicle or morphine (2 mg/Kg, i.p.) and the preference was immediately measured by giving the animals free access to both sides of the apparatus for 15 min (Gawel et al., 2014).

Table 1 Fatty acids composition of the different enriched diets (% of total fatty acids identified). Constituents

MBD

WBD-PO

WBD-IF

∑SFA C16:0 ∑MUFA ∑n-3 PUFA ∑n-6 PUFA n-6/n-3 PUFA

30.47 19.80 26.68 14.66 28.19 1.92

44.63 38.38 42.59 0.49 12.29 25.24

52.24 35.23 33.36 0.69 13.70 19.78

Abbreviations: MBDMediterranean-based diet; WBDWestern based diet; POpalm oil; IFinteresterified fat; SFASaturated fatty acids; MUFAMonounsaturated fatty acids; PUFAPolyunsaturated fatty acids.

iii) IF (rich in SFA Interesterified) (Triângulo Alimentos®, São Paulo, Brazil). Diets were weekly prepared during the experimental protocol, stored at -20 °C in daily portions (Magri et al., 2014) and given to the animals every 24 h. 2.4. Dietary fatty acids

2.7. Elevated plus maze (EPM) task

The diets fat (Table 1) was extracted with chloroform-methanol according to Bligh and Dyer (1959) followed by saponification in methanolic KOH solution and esterification in methanolic H2SO4 solution (Hartman and Lago, 1973). Methyl ester fatty acids were analyzed using an Agilent Technologies gas chromatograph (HP 6890) equipped with a DB-23 capillary column (60 m x0.25 mm x0.25 μm) and flame ionization detector. The temperature of the injector port was set at 250 °C and the carrier gas was nitrogen (0.4 mL/min). After injection (1 μL, split ratio 50:1), the oven temperature was hold at 180 °C for 3 min, then it was increased to 200 °C at 10 °C/min and this temperature was kept for 20 min. In sequence, it was increased to 240 °C at 9 °C/min and this temperature was kept for 15 min. Standard fatty acid methyl esters (37component FAME Mix, C 22:5n3 and PUFA no. 2 from Sigma, Saint Louis, MO, USA) were run under the same conditions and the subsequent retention times were used to identify the fatty acids. Fatty acids were expressed as percentage of the total ones identified.

Anxiety index values range from 0 to 1, where increased values indicate increased anxiety-like behavior (Cohen et al., 2008).

2.5. Drugs and solutions

2.8. Locomotor activity measurement

Morphine sulfate (Cristália®, São Paulo, Brazil) was diluted in 0.9% NaCl solution and intraperitoneally injected (i.p.) in a dose of 4 mg/kg for conditioning (Vey et al., 2015; Roversi et al., 2016; Milanesi et al., 2017), 2 mg/kg for reinstatement (adapted from Assar et al., 2016) and vehicle injections were 0.9% NaCl solution in a 1 mL.

Spontaneous locomotor activity of individual rats was evaluated in Open Field task (OF) during morphine withdrawal. In this test the rats were individually placed in the center of an open field arena (40 × 40 × 30 cm) divided in nine quadrants. The number of square crossings and rearing was recorded for 5 min (Kabuki et al., 2009) and used as measures of spontaneous locomotor activity and exploratory behavior, respectively.

To assess the influence of different enriched diets on anxiety-like symptoms during morphine withdrawal, animals were observed in the EPM, which is based on the innate fear that rodents have for open and elevated spaces (Montgomery, 1955). The apparatus was allocated in a room with low light intensity, presenting a cross shape, with two open arms (50 cm × 10 cm) facing each other; and two arms of the same dimensions closed by side walls and opposite to each other. All arms have a central intersection (10 cm × 10 cm). On the test day, animals were individually placed in the central intersection facing an open arm. The number of entries and the time spent in the open arms were quantified for 5 min in EPM. The anxiety index was calculated as described in the following formula: Anxiety index= 1 −

2.6. Conditioned place preference (CPP) procedure

((

Openarmstime totaltime

)+(

Openarmsentries Totalentries

)/2)

2.9. Thermal sensitivity assessment with the hot plate test

The CPP procedure is a well-established behavioral paradigm that has been widely employed to assess symptoms of reward, extinction and relapse to addictive drugs (Bardo and Bevins, 2000; Gawel et al., 2014). It uses three compartments in the boxes, which are separated by manual guillotine doors: two compartments of equal size (45 cm × 45 cm × 50 cm) with different visual clues: one with white floor and striped walls and other with striped floor and smooth white walls. These two compartments converge to a third smaller compartment. The CPP procedure were performed following these steps: habituation, pre-test, conditioning, test, extinction and reinstatement test. On day 1, rats were kept for 15 min in each compartment for habituation. On the next day, we performed the pre-test, that consists of letting the animal freely choose one of the compartments for 15 min. Animals that showed preference (more than 70%) for any compartment were excluded from the experimental protocol. On the following 4 days, the animals were conditioned with morphine (4 mg/kg, i.p.) and placed for 45 min in the compartment they spent less time during the pre-test, and then with the

Thermal sensitivity was assessed in the hot plate test during morphine withdrawal according to a previously described method (Woolfe and MacDonald, 1944). In this test, animals were placed in the hot plate apparatus (Insight® EFF- 361), and a hot thermal stimulus on a metal plate (52 °C ± 0.5 °C) was used to determine their thermal sensitivity. The latency for removal or licking the paws was recorded, where lower latency corresponds to increased thermal sensitivity. If animals did not respond within 30 s (cut-off time), they were removed from the plate to avoid tissue damage. After the hot plate test, animals were placed back in the experimental cages (Haleem and Nawaz, 2017). 2.10. Immunoblotting Brain areas were removed according to Paxinos and Watson (2013). PFC and NAc were homogenized with a lysis buffer containing 137 mM 9

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Two-way ANOVA of CORT showed a significant main effect of the diet and drug [F(2,24) = 110.3 and 86.2, p = 0.000, respectively]. Regardless of the diet, Post hoc analysis showed that WBD-PO and WBDIF groups had their CORT plasma levels increased when compared to MBD, while morphine-reinstatement increased this blood parameter in all groups. (Table 2). Two-way ANOVA of ACTH showed a significant main effect of the diet, drug and diet x drug interaction [F (2,24) = 231.5 and 117.4, p = 0.000 and 8.1, p = 0.001, respectively]. Regardless of the diet, Post hoc analysis showed that WBD-PO and WBDIF groups increased the plasma levels of ACTH in relation to MBD. Morphine-reinstatement increased this parameter in all groups (Table 2).

NaCl, 20 mM Tris–HCl pH 8.0, 1% NP40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg mL-1 aprotinin, 0.1 mM benzethonium chloride, and 0.5 mM sodium orthovanadate. Protein determination was performed in the supernatant after sample centrifugation according to the MicroBCA procedure (Pierce, IL, USA). Proteins were separated by electrophoresis on 10% and 12% sodium dodecyl sulfate-polyacrylamide gels (SDS–PAGE), and electrotransferred to PVDF membranes (Milipore, MA, USA). Non-specific binding sites were blocked in Tris-buffered saline (TBS), pH 7.6, containing 5% non-fat dry milk. Membranes were rinsed in buffer (0.05% Tween-20 in TBS) and then incubated with primary antibodies: anti-GR (1:1000), anti-β actin (1:2000), anti-DAT (1:1000), anti-D2R (1:3000), anti-D1R (1:500) (Santa Cruz Biotechnology, Santa, Cruz, CA, USA) followed by anti-rabbit or anti-goat IgG horseradish peroxidase conjugate (1:40.000; Santa Cruz Biotechnology). After being rinsed with buffer, immune complexes were visualized by chemiluminescence using the ECL kit (Amersham Pharmacia Biotech Inc., NJ, USA), and band intensities were quantified with ImageJ software (NIH). Actin was used as an internal control and data were standardized according to actin values.

3.2. Assessment of the development of morphine preference in conditioned place preference (CPP) Two-way ANOVA of CPP-test revealed a significant main effect of drug and diet x drug interaction [F(2,36) = 66.9, p = 0.000 and 5.0, p = 0.01, respectively]. Post hoc showed that MBD-, WBD-PO and WBDIF groups spent longer time in the drug compartment, as expected, indicating morphine-CPP. Interestingly, among morphine-conditioned animals, WBD-IF group showed more preference than the MBD group, whereas the WBD-PO was not different from any of them (Fig. 2).

2.11. Plasma corticosterone (CORT) and Adrenocorticotrophic hormone (ACTH) measurement Blood aliquots were centrifuged at 4◦C at 1500 × g for 15 min. Plasma aliquots were frozen at -80◦C until their analysis. CORT and ACTH plasma levels were analyzed through enzyme immunoassay (ELISA) using a commercial kit following the manufacturer’s instructions (LDN - Labor Diagnostika Nord and Sigma-Aldrich®, respectively).

3.3. Assessment of the anxiety-like behaviors in the elevated plus maze (EPM) task during drug withdrawal Two-way ANOVA of EPM revealed a significant main effect of the diet, drug and diet x drug interaction [F(2,36) = 6.25, p = 0.004, 47.3 and 8.4, p = 0.000, respectively] on time spent in open arms and [F (2,36) = 4.0, p = 0.025; 19.9, p = 0.000 and 6.4, p = 0.004 respectively] on number of open arms entries. Two-way ANOVA of EPM revealed a significant main effect of the drug [F(2,36) = 24.1, p = 0.000] on the anxiety index. Post hoc test showed that animals fed with both WBD-PO and WBDIF presented decreased time spent and entries number per se in the open arms in the EPM in relation to MBD (Fig. 3A and 3B). Morphine conditioning decreased the time spent in the open arms during its withdrawal, without considering the diet, thus indicating abstinence symptoms development (Fig. 3A). In addition, MBD group showed decreased entries number in the open arms during morphine withdrawal, while in WBD-PO and WBD-IF, this behavioral parameter was similar to their pairs that were not conditioned with morphine (Fig. 3B). In the same way, the anxiety index was higher per se in WBD-PO and WBD-IF groups, while this index was increased in all experimental groups during morphine abstinence, regardless of the diet (Fig. 3C).

2.12. Statistical analysis Two-way ANOVA followed by Duncan’s test was performed (Software package Statistic 8.0 for Windows). All data are expressed as means ± standard error of the mean (S.E.M.). Values of p < 0.05 were considered statistically significant for all comparisons made. GraphPad Prism® (version 5.01) was used to create the figures. 3. Results 3.1. Body weight, epididymal fat (epididymal fat weight/body weightEFW/BW), adrenal weight (adrenal weight/body weight- AW/BW), corticosterone (CORT) and adrenocorticotrophic hormone (ACTH) plasma levels The experimental groups showed no difference in body weight (Table 2). Two-way ANOVA of EFW/BW and AW/BW showed a significant main effect of the diet [F(2,36) = 22.3 and 17.3, p = 0.000, respectively]. Post hoc analysis showed that both WBD-PO and WBD-IF increased EFW/BW and AW/BW in comparison to MBD (Table 2).

Table 2 Influence of different enriched diets and morphine reinstatement on epidydimal fat weight/body weight, adrenal weigh/body weight, CORT and ACTH plasma levels. Groups

Body Weight (g)

EFW/BW Ratio

AW/BW Ratio

CORT (ng/ml)

398.7 ± 15.9 399.5 ± 14.5 390.1 ± 11.8

1.50 ± 0.06b 2.12 ± 0.11a 2.37 ± 0.04a

0.11 ± 0.00 0.16 ± 0.00 0.15 ± 0.01

b

391.0 ± 15.4 397.8 ± 13.4 369.2 ± 13.3

1.75 ± 0.10 2.19 ± 0.13 2.11 ± 0.10

0.11 ± 0.00 0.15 ± 0.01 0.15 ± 0.00

b

ACTH (ng/ml)

Vehicle MBD WBD-PO WBD-IF Morphine MBD WBD-PO WBD-IF

b a a

a a

a a

101.8 ± 4.5 158.0 ± 5.0 151.2 ± 6.4

b

129.6 ± 1.8 203.0 ± 3.3 183.1 ± 1.1

*b

a a

*a *a

61.4 ± 3.8 c 135.0 ± 3.8 a 81.6 ± 4.2 b 83.2 ± 2.8 *c 158.4 ± 3.1 *a 128.4 ± 2.6 *b

Abbreviations: MBD: Mediterranean-based diet; WBD: Western based diet; PO: palm oil; IF: interesterified fat; BW: body weight; EFW: Epidydimal fat weight; AW: Adrenal weight. CORT: corticosterone; ACTH: Adrenocorticotrophic hormone. *indicates significant difference among treatments in the same diet; Different lowercase indicates significant difference among diets in the same treatment, where "a" is different from "b" and "ab" is equal to both. 10

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3.6. Influence of different enriched diets on morphine reinstatement assessed in CPP Two-way ANOVA of morphine-reinstatement revealed a significant main effect of the diet, drug and diet x drug [F(2,36) = 5.85, p = 0,006; 31.62, p = 0,000 and 3.65, p = 0,035, respectively]. Post hoc analysis revealed that morphine-reinstatement increased the time spent in the drug-conditioned place after its extinction in WBD-PO and WBD-IF groups (38.7% and 46.8%, respectively) in comparison to MBD (11.1%), which did not show morphine-reinstatement. In fact, among the vehicle-injected groups the time spent in both sides of CPP was the same, indicating no place preference (Fig. 6). Fig. 2. Influence of different enriched diets on morphine induced conditioned place preference (CPP). Data are expressed as mean ± S.E.M. *indicates significant difference among treatments in the same diet; Different lowercase indicates significant difference among diets in the same treatment, where "a" is different from "b" and "ab" is equal to both; (p < 0.05). Abbreviations: MBD: Mediterranean-based diet; WBD: Western based diet, PO: palm oil; IF: interesterified fat.

3.7. Influence of different enriched diets and morphine reinstatement on the glucocorticoid receptors (GR) immunoreactivity in the prefrontal cortex (PFC) Two-way ANOVA revealed a significant main effect of the diet and drug in PFC [F(2,24) = 13.92, p = 0.000 and 10.70, p = 0.003, respectively] on GR immunoreactivity. Post hoc test showed that WBD-PO and WBD-IF groups increased GR immunoreactivity in comparison to MBD and morphine reinstatement increased the level of this molecular parameter in WBD-IF group when compared to its vehicle (Fig. 7A). Interestingly, two positive linear correlations between plasma level of CORT with both GR immunoreactivity (R2 = 0.56, p = 0.000) and AW/BW ratio (R2 = 0.43, p = 0.000) were observed (Fig. 7B and C).

3.4. Assessment of the locomotor and exploratory performance in the open field (OF) task Two-way ANOVA of the OF task revealed a significant main effect of the diet, drug and diet x drug interaction [F(2,36) = 9.3 p = 0.0005, 13.0, P = 0.0009 and 4.4, p = 0.0204, respectively] on the crossing number as well as a significant main effect of diet and drug [F (2,36) = 9.9, P = 0.003 and 14.0, p = 0.0006, respectively] on the rearing number. Post hoc test showed that WBD-PO and WBD-IF groups showed a smaller crossing number per se in relation to MBD, and no difference of rearing number was observed. Furthermore, after the morphine withdrawal, WBD-PO and WBD-IF groups showed increased crossing- and decreased rearing- number in comparison to their vehicle injected pairs (Fig. 4A and 4B).

3.8. Influence of different enriched diets and morphine reinstatement on immunoreactivity of dopaminergic targets in the NAc Two-way ANOVA revealed a significant main effect of the diet, drug and diet x drug interaction in the NAc [F(2,24) = 5.92, p = 0.008, 50.73, p = 0.000 and 6.87, p = 0.004, respectively] on DAT, [F (2,24) = 6.98, p = 0.004, 37.66, p = 0.000 and 5.44, p = 0.01, respectively] on D1R and [F(2,24) = 16.68, p = 0.000, 45.34, p = 0.000 and 7.37, p = 0.003, respectively] on D2R immunoreactivity. The different diets exerted no influence per se on DAT, D1R or D2R in NAc (Fig. 8A, B, C). Among morphine conditioned animals, the drug reinstatement increased DAT and D2R (Fig. 8A, C) and decreased D1R (Fig. 8B) levels in WBD-PO and WBD-IF but not in MBD.

3.5. Thermal sensitivity assessment with the hot plate test Two-way ANOVA of the hot plate test revealed a significant main effect of the diet [F(2,36) = 4.7, p = 0.015]. Post hoc test showed that during the morphine withdrawal, WBD-IF group showed lower latency time to paw removal when compared to its vehicle and the MBD fed group, consequently indicating thermal hyperalgesia development. In fact, after morphine withdrawal, the MBD and WBD-PO showed a comparable response in this behavioral paradigm (Fig. 5).

4. Discussion A major obstacle to prevent the reinstatement in addicts is to understand how different life experiences can lead to this common end point (Ebner et al., 2018). In this sense, long-lasting vulnerability to reinstatement has been recognized as a pivotal phenomenon for the understanding and treatment of drug addiction (See, 2005). During abstinence, drug relapse is often precipitated by acute re-exposure to

Fig. 3. Influence of different enriched diets on anxiety-like behaviors in the elevated plus maze (EPM) task during morphine withdrawal. (A) Number of entries in the open arms; (B) Time spent in the open arms; (C) Anxiety index. Data are expressed as mean ± S.E.M. *indicates significant difference among treatments in the same diet; Different lowercase indicates significant difference among diets in the same treatment, where "a" is different from "b" and "ab" is equal to both; (p < 0.05). Abbreviations: MBD: Mediterranean-based diet; WBD: Western based diet, PO: palm oil; IF: interesterified fat. 11

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Fig. 4. Influence of different enriched diets on locomotor and exploratory activity during morphine withdrawal. A) Number of crossing; B) Number of rearing. Data are expressed as mean ± S.E.M. *indicates significant difference among treatments in the same diet; Different lowercase indicates significant difference among diets in the same treatment, where "a" is different from "b" and "ab" is equal to both; (p < 0.05). Abbreviations: MBD: Mediterranean-based diet; WBD: Western based diet, PO: palm oil; IF: interesterified fat.

observed in plasma CORT and ACTH levels and GR immunoreactivity. Furthermore, our findings also showed that all diets were able to induce morphine preference in CPP paradigm, consequently increasing anxiety-like behaviors after the drug discontinuation, thus reflecting abstinence symptoms; however, during abstinence only the Western based diets iv) facilitated locomotor sensitization v) induced thermal hypersensitivity vi) activated HPA axis. The Western based diets showed morphine-reinstatement and modified DA neurotransmission unlike the Mediterranean-based diet. The chronic consumption of the diets caused no differences in the body weight; however, both Western based ones favored a visceral fat gain, since the epididymal fat weight/body weight ratio was increased (Nagy et al., 2018) in the WBD-PO and WBD-IF. Interestingly, adrenal weight/body weight ratio in both Western based diets was higher than those observed in Mediterranean-based diet. In this context, it has already been described that dietary FA can modulate the HPA axis functioning and consequently the adrenal weight (Bose et al., 2009). In fact, FA are freely transported across the blood-brain barrier (Dhopeshwarkar and Mead, 1973), making the brain FA homeostasis dependent on plasma free FA (FFA) levels. This way, elevated plasma FFA levels can exacerbate cellular damage and affect brain functions (Adibhatla and Hatcher, 2008). Interestingly, alterations in plasma FFA, including plasma levels of the SFA palmitic acid, which is present in both WBD, has been reported to stimulate the HPA axis (Hryhorczuk et al., 2017). Such HPA axis stimulation is related to the increased adrenal weight (Gouirand and Matuszewich, 2005) and higher secretion of adrenocorticotrophic hormone (ACTH), consequently elevating the glucocorticoids blood levels (Cox et al., 2011). Regarding this, our outcomes showed that the WBD-PO and WBD-IF were able to increase CORT and ACTH plasma levels per se and after morphine reinstatement. Increased plasma level of glucocorticoids is frequently related to behavioral changes linked to anxiety (Lee et al., 2008), whose discommodity may be a factor in the reinstatement of abused substances and addiction (Nimitvilai et al., 2014). Corticosterone, the major stress hormone, is released from the adrenal cortex acting as a neuromodulator in extrahypothalamic brain areas including the prefrontal cortex (PFC) through binds to glucocorticoid receptor (GR) (McEown and Treit, 2011). GRs are widely expressed in the brain, particularly in limbic structures such as the PFC, and its role is closely involved in the anxiety-like behaviors. (LeDoux, 2000). Larrieu et al. (2016) demonstrated that GR-mediated signaling pathway in PFC was negatively affected by the n-3 PUFA deficiency diet. Additionally, our findings showed increased GR immunoreactivity in the PFC in the same animals that presented adrenal weight gain, i.e, in WBD fed groups. Besides that, we observed a positive linear correlation of the corticosterone plasma level with the GR immunoreactivity in PFC and AW/BW ratio, suggesting that the chronic consumption of foods rich in PO and/ or IF, frequently observed in Western Countries, may adversely modulate the HPA axis responses. Literature has shown that increased sensitivity to stress has been

Fig. 5. Influence of different enriched diets on latency response for thermal stimuli assessed in the hot plate (HP) test during morphine withdrawal. Data are expressed as mean ± S.E.M. *indicates significant difference among treatments in the same diet; Different lowercase indicates significant difference among diets in the same treatment, where "a" is different from "b" and "ab" is equal to both; (p < 0.05). Abbreviations: MBD: Mediterranean-based diet; WBD: Western based diet, PO: palm oil; IF: interesterified fat.

Fig. 6. Influence of different enriched diets on morphine reinstatement in conditioned place preference apparatus. Data are expressed as mean ± S.E.M. *indicates significant difference among treatments in the same diet; Different lowercase indicates significant difference among diets in the same treatment, where "a" is different from "b" and "ab" is equal to both; (p < 0.05). Abbreviations: MBD: Mediterranean-based diet; WBD: Western based diet; PO: palm oil; IF: interesterified fat.

drug-associated cues, stress or neuroadaptive dysregulation previously induced by the chronic drug use (Shaham and Hope, 2005). According to this and regarding this study, for the first time it was observed that both WBD-PO and WBD-IF had their behavioral biochemical and molecular parameters modified by themselves. The diets were able to: i) increase anxiety-like symptom as observed by the decreased time spent and entries number in the open arms of the EPM together with increased anxiety index; ii) decrease locomotion activity as observed by the reduced crossing number in the OF task; iii) activate HPA axis as 12

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Fig. 7. Influence of different enriched diets and morphine reinstatement on A) immunoreactivity of glucocorticoid receptors in prefrontal cortex; B) Correlation between GR immunoreactivity and CORT; C) Correlation between AW/BW ratio and CORT. Data are expressed as mean ± S.E.M. *indicates significant difference among treatments in the same diet; Different lowercase indicates significant difference among diets in the same treatment, where "a" is different from "b" and "ab" is equal to both; (p < 0.05). Abbreviations: MBD: Mediterranean-based diet; WBD: Western based diet; PO: palm oil; IF: interesterified fat; GR: glucocorticoid receptor; CORT: corticosterone; AW/BW: adrenal weight/body weight ratio.

PUFA diet significantly increased depressive-like behavior, which was related to increased plasmatic Aβ levels (Bove et al., 2018). In fact, depression is more frequently observed in the female gender, which shows greater response to stress consequently favoring opioid abuse when compared to the male gender (Chartoff and McHugh, 2016). Severe pain often leads to stress, anxiety, depression and other neuropsychiatric comorbidities, facilitating opioid misuse or abuse in individuals without other risk factors (Webster, 2017). As expected, our study showed that following the morphine withdrawal all experimental groups showed anxiety-like symptoms but only animals fed with both WBD-PO and WBD-IF presented locomotor changes, as observed in the OF task. Locomotor sensitization is also a behavior that deserves to be considered to evaluate addiction-like behavior as it is well established that repeated drug administration leads to increased locomotor activity (Melchiorri et al., 1992). As it was already described, in our outcomes the consumption of both WBD reduced locomotor activity and this was not observed in the MBD fed animals. This finding is in agreement with literature when the consumption of a SFA rich diet, especially palmitic acid, decreased the locomotor activity of the animals, which number was negatively correlated with an increased n-6/n-3 ratio in the brain (Moon et al., 2014). However, an increased locomotor activity in WBD groups, but not in

connected to anxiety-like behavior (Aston-Jones and Harris, 2004). Considering this, as observed in our EPM paradigm, consumption of both Western based diets was related to increased GR and adrenal weight and increased anxiety behaviors, which were not observed in Mediterranean-based diet fed animals. These findings agree with literature data that have shown increased anxiety induced by both palatable diets and high fat diet (Sharma et al., 2013; Moon et al., 2014). In line with our findings, recent studies show that the consumption of n-3 PUFA enriched foods is related to reduced anxiety in preclinical (Pusceddu et al., 2015; Hakimian et al., 2017) and clinical trials (Lesperance et al., 2011), leading us to suggest that the anxiogenic response observed in animals fed with both Western based diets was due to the low n-3 levels and high n-6/n-3 ratio. It is well known that anxiety symptoms constitute a pivotal drug withdrawal symptom in humans (Koob and LeMoal, 2005) and it is also observed in experimental studies (Zhang and Schulteis, 2008). Considering anxiety-like symptoms, our current findings are in agreement with the literature. Morgese et al. (2017) showed a relation between the dietary consumption of n-6 PUFA and increased anxietyand depressive-like behaviors in rats. Inversely, a high dietary n-3 PUFA intake decreased such behaviors (Morgase et al., 2017). In agreement with this finding, Bove et al., showed that female rats fed with poor n-3

Fig. 8. Influence of different enriched diets and morphine reinstatement on dopaminergic targets in nucleus accumbens A) DAT; B) D1R; C) D2R. Data are expressed as mean ± S.E.M. *indicates significant difference among treatments in the same diet; Different lowercase indicates significant difference among diets in the same treatment, where "a" is different from "b" and "ab" is equal to both; (p < 0.05). Abbreviations: MBD: Mediterranean- based diet; WBD: Western based diet; PO: palm oil; IF: interesterified fat; D1R: Dopaminergic 1 receptor; D2R: dopaminergic 2 receptor; DAT: dopamine transporter. 13

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and increasing D2R in NAc (Naneix et al., 2017). In agreement with this, Burke and Miczek (2014) suggested a possible sensitization of the mesolimbic DA system as possible neural mechanisms for addiction, which occur together with an increased DA content in the NAc Therefore, we hypothesize that the higher reinstatement rates observed in both Western based diets groups, induce greater DA release in NAc, as well as increased DAT, D2R upregulation and D1R desensitization. In summary, our study provides evidences that the chronic consumption of Western diets enhances the mesolimbic DA system sensitivity, favoring withdrawal symptoms and reinstatement to opioid addiction. Previous studies from our laboratory have shown that trans fat was incorporated into neural membrane phospholipids, increasing additive properties morphine- and amphetamine- induced in both CPP and self-administration paradigms, respectively (Roversi et al., 2016; Kuhn et al., 2015a, b). The present study highlights evident neurobiological mechanisms which could support the high reinstatement incidence to drug use after the abstinence period, especially opioid drugs. Based on this, some hypotheses deserve to be considered: i) Western diets can exert deleterious influences on the HPA axis, so facilitating anxiety-like symptoms development; ii) Western diets are able to enhance the mesolimbic DA system sensitivity to addictive drugs such as morphine.

MBD, during morphine withdrawal was observed, suggesting locomotor sensitization characteristic of addiction-like behavior. Repeated administration of addictive drugs, such as morphine, is known to increase locomotor activity as a development of locomotor sensitization and this has been suggested as one of the addiction-like behaviors observed in rodents (Robinson and Berridge, 2003). In the same way, WBD groups during morphine withdrawal showed a reduction in natural exploratory behavior shown by decreased rearing activity. Rearing is considered an exploratory behavior evoked by novel stimuli and it is an important aspect of the individual behavior because it increases the knowledge about the environment and thereby increases the chances of finding food, mates and shelter (Alves et al., 2012). The prolonged use of opioids is related to tolerance development, while the activation of pronociceptive mechanisms leading to increased pain sensitivity (hyperalgesia) that are related to the withdrawal syndrome (Hutchinson et al., 2011). In this way, our findings showed that during morphine withdrawal, both Western based diets, containing low n-3 PUFA content, showed hyperalgesia development, as observed in the reduced paw withdrawal latency time. Interestingly, a recent study of our research group (Milanesi et al., 2017) showed that IF supplementation increased the thermal sensitivity of female rats in the HP test. On the other hand, Escudero et al. (2015) reported that n-3 supplementation attenuated morphine tolerance development in HP test, while Nakamoto et al. (2012) show that these n-3 FA were able to facilitate the β-endorphin releasing, evocating an antinociceptive activity. All addictive drugs, directly or indirectly, are capable to intensify DA release in the mesolimbic structures, leading to a positive reinforcement, which may be observed by CPP development (Tzeng et al., 2015). Morphine, which is an agonist drug of mμ opioid receptor, inhibits GABAergic neurons in the ventral tegmental area (VTA), increasing DA release in the NAc, which expresses D1 and D2 receptors (Listos et al., 2013). At least two types of proteins participate in the regulation of the DA extracellular concentration addictive drugs induced, DAT and D2R. Furthermore, in the pre-synaptic dopaminergic nerve terminal, DAT re-uptakes DA from the synaptic cleft into DAergic neurons (Giros et al., 1996). Mesolimbic dopaminergic neurons have D2R that act as autoreceptors to inhibit spontaneous firing, as well as DA release, when activated by extracellular DA (Rouge-Pont et al., 2002). Dopaminergic neurotransmission through D1R is able to control the acquisition of opioid reward memory, whose activation has been experimentally related to initial morphine preference (Lintas et al., 2011; Narita et al., 2005). D1R and D2R signaling are differently involved in the acquisition and retrieval of morphine contextual memory, since activation of D1R is necessary for morphine-CPP expression and D2R activation is necessary for morphine-CPP reinstatement (Wang et al., 2018). Apparently, the role of D2R on morphine-reinstatement seems be more significant than that of the D1R (Assar et al., 2016). In this way, Farahimanesh et al. (2018) demonstrated that D2R antagonists in VTA attenuated morphine reinstatement, while Sadeghzadeh et al. (2017) reported that D2R antagonist in NAc attenuated the morphine-reinstatement with ineffective morphine dose plus food deprivation. Our current findings demonstrated that both Western based diets increased morphine-reinstatement following abstinence period what was not observed in MBD fed animals. Therefore, after morphine-reinstatement both WBD-PO and WBD-IF showed increased D2R and DAT together with decreased D1R in NAc, unlike the MBD that showed no such changes after morphine-reinstatement. From these findings we can infer that greater DAT immunoreactivity was due to a greater DA release in the synaptic cleft, which activated D2R autoreceptors to reduce the dopaminergic signaling. In the same way, a decreased D1R imunoreactivity may happen because of its desensitization by the higher DA presence in the synaptic cleft. Literature data have shown that palatable food enhances sensitivity of the mesolimbic DA system though increasing DA neurons activity, DA release

5. Conclusion This study evaluates factors that may facilitate the morphine addition and behaviors that can lead to the reinstatement to opioid drugs. In this sense, we show that Western based diets, which are rich in SFA and low n-3 PUFA, can facilitate morphine reinstatement. The current outcomes propose that increased dietary intake of n-3 PUFA exerts beneficial influences on anxiety disorders, including drug addiction. We hope that the present study may alert health authorities around the world about trans fat replacement by the interesterified and palm oil. The chronic consumption of these two fats may hinder detoxification treatments in opioid drugs what is a current and serious public health problem in different countries. Conflict of interest The authors declare no conflict of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001. Authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasil); CAPES, Brasil; Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, Brasil) and PRPGP-UFSM (PROAP) for their fellowships and financial support. The authors thank Triângulo Alimentos® for the donation of interesterified fat, Laboratório Tiaraju® for the donation of n-3 and Agropalma® for the donation of palm oil. References Adibhatla, R.M., Hatcher, J.F., 2008. Altered lipid metabolism in brain injury and disorders. Subcell. Biochem. 49, 241–268. https://doi.org/10.1007/978-1-4020-88315_9. Alves, R., Barbosa de Carvalho, J.G., Benedito, M.A., 2012. High and low rearing subgroups of rats selected in the open field differ in the activity of K+-stimulated pnitrophenylphosphatase in the hippocampus. Brain Res. 1058 (1-2), 178–182. https://doi.org/10.4236/jbbs.2012.23036.

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L.H. Milanesi, et al.

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