Valenza M. Et Al. Reward Deficits in Obesity-Prone Rats
Valenza M. et al. Reward deficits in obesity-prone rats
Diet-Induced Obesity and Diet-Resistant rats: differences in the rewarding and anorectic effects of D-amphetamine
Marta Valenza, Ph.D. 1,2,†, Luca Steardo, M.D. 3, Pietro Cottone, Ph.D. 1*, Valentina Sabino, Ph.D. 1*
1 Laboratory of Addictive Disorders, Departments of Pharmacology and Psychiatry, Boston University School of Medicine, Boston, MA; 2 Department of Biomedical Sciences and Human Oncology, Section of Pharmacology, University of Bari Aldo Moro, Bari, Italy; 3 Department of Physiology and Pharmacology, Sapienza University of Rome, Rome, Italy.
* These authors contributed equally to this work
†Current address: Laboratory of the Biology of Addictive Diseases, The Rockefeller University, New York, NY
Correspondence and requests for materials should be addressed to:
Valentina Sabino (email: ) or Pietro Cottone (email: )
Laboratory of Addictive Disorders
Departments of Pharmacology and Psychiatry
Boston University School of Medicine
72 E Concord St, R-612 or R-618, Boston, MA 02118 USA
Phone: 617-638-4327 or 617-638-5662
Fax: 617-638-5668
Supplemental Materials and Methods
Subjects
Diet-Induced Obesity (DIO) and Diet Resistant (DR) rats (n=44) were purchased from Taconic Farms (TacLevin: CD(SD)DIO and CD(SD)DR, Taconic Farms, Inc., Huston, NY, USA)(Levin et al. 1997). Rats, weighing 325-350g upon arrival, were housed in a 12h reverse light/dark cycle (lights off at 11AM), in an AAALAC-approved humidity- and temperature-controlled vivarium. In “pre-obesity” DIO and DR rats were fed ad libitumvivarium chow, a corn-based Harlan Teklad LM-485 Diet 7012 (65% [kcal] carbohydrate, 13% fat and 21% protein; metabolizable energy 3.1 kcal/g; Harlan - Indianapolis, IN, USA)(Cottone et al. 2013; Cottone et al. 2007). In “obesity” DIO and DR rats were fedad libitum the high-fat diet D12266B Research Diets (51.4% [kcal] carbohydrate, 31.8% fat and 16.8% protein; metabolizable energy 4.41 kcal/g; Research Diets, Inc. - New Brunswick, NJ, USA). Rats from both groups were fed the D12266B high-fat diet for at least 4 weeks before starting thesecond part of the experiments, whichallowedDIO rats to develop obesity.(Ricci and Levin 2003)Tests were performed during the dark cycle of the day (rodents’ active phase).
All procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Principles of Laboratory Animal Care and were approved by the Institutional Animal Care and Use Committee of Boston University Medical Campus.
Drugs
D-amphetamine hemisulfate salt C-II (Sigma-Aldrich, St. Louis, MO) was dissolved in sterile isotonic saline. Doses (0, 0.1, 0.5 and 1 mg/kg) were administered intraperitoneally (1ml/kg) 10 minutes before each experiment,usinga Latin squarewithin-subject design.(Esposito et al. 1980; Grilly and Loveland 2001)Drug treatments were separated at least by 1-2 treatment-free days until variables returned to baseline.
Surgery for electrode implantation
Surgery for electrode implantation was performed as previously described.(Dore et al. 2013a; Iemolo et al. 2012)DIO and DR rats were anesthetized with isoflurane (2-3% in oxygen) and secured in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). One 0.125 mm diameter bipolar stainless steel electrode (MS303/3-B/SPC, length 10.5 mm; Plastics One, Roanoke, VA) was unilaterally implanted to the left or right lateral hypothalamus (LH)using the following coordinates: AP -0.5 mm andML ±1.7 mm from the bregma, , DV -9.7 mm from skull, with the incisor bar set 5.0 mm above the interaural line, according to Pellegrino’s atlas.(Pellegrino et al. 1979) Four stainless steel jeweler’s screws were fastened into the rat’s skull around the electrode. Dental restorative filled resin (Henry Schein Inc., Melville, NY, USA) and acrylic cement were applied forming a pedestal that firmly anchored the electrode in place.
Apparatus for intracranial self-stimulation
ICSS training and testing took placeaspreviously described in modular operant chambers operant test chambers (30×24×29 cm) (Med Associates Inc., St. Albans, VT).(Iemolo et al. 2012)Chambers had a grid floor and were located in ventilated, sound-attenuating enclosures (66×56×36 cm). Each chamber had a retractable lever on one side wall(Cottone et al. 2012).Subjects were connected to the electrical stimulation circuit by bipolar leads (Plastics One) and gold contact swivel commutators (Plastics One). Constant current square wave stimulators (Med Associates) were used to deliver electrical brain stimulation. A computer with a 10-ms resolution controlled all programming functions.
Intracranial self-stimulation procedure
The ICSS procedure was performed following a previously published procedure.(Dore et al. 2013a; Iemolo et al. 2012)Rat reward thresholds were assessed using a rate-independent, discrete-trial, current intensity procedure originally designed by Kornetsky and colleagues.(Esposito and Kornetsky 1977; Marcus and Kornetsk.C 1974)After recovery from surgery, rats were trained to lever press on a fixed ratio 1 (FR1) schedule of reinforcement to obtain an electrical stimulation. Each stimulus consisted of a 500 ms train with a pulse width of 0.2 ms and a delay of 0.2 ms between the positive and negative pulses.
Once stable FR1 operant responding was established, ICSS thresholds were assessed using the following procedure:at the beginning of each trial, rats received a non-contingent stimulus (S1), after which they had the opportunityto lever press during a 7.5 s period. The lever press resulted in the delivery of a contingent stimulus (S2) that was identical to the previous S1. A 7.5–22.5 s (average 15 s) period of time elapsed between S2 delivery and the delivery of the next S1. If no response occurred, this time period began at the end of the 7.5-s period allotted for response. These time periods were randomized so that animals could not predict the next S1 delivery. A trial consisted of five presentations of S1 at a fixed current intensity (in μA). Three or more responses at that intensity were scored as a plus (+), whereas two or fewer responses were scored as a minus (−) for that trial.If the animal scored a (+) for the first trial, the second trial began at an intensity of 5 μA lower than the first. The current intensity continued to decrease by the same fixed intensity until the animal scored a (−) for two consecutive trials. When this occurred, the current intensity of the second trial with a (-) score was repeated. The current intensities were then ascended by 5 μA for each trial until the animal scored a (+) for two consecutive trials.
Each set of ascending or descending current intensities was defined as a “column”, and a total of six alternating descending/ascending columns were included in each session. The intensity at the midpoint between (+) and (−) was defined as the “column threshold”. The rat ICSS threshold for each session was calculated as the mean of the last four column thresholds. The reward threshold is, therefore, the minimal current intensity able to produce a response that maintains the self-stimulation behavior.(Markou and Koob 1991)
A raise in the reward threshold indicated that stimulus intensities that were previously perceived as reinforcing were no longer perceived as rewarding, reflecting a decrease in reward function and suggesting a depressive-like state. Conversely, lowering of the reward threshold reflected increased reward function.
To discourage the subject from responding during the inter-trial interval (ITI), any response during this period postponed the onset of the S1 for an additional 22.5 s (a length of time that exceeded or was equal to the original random duration of the ITI). These “punished” responses were recorded as timeout responses, and represented a measure of impulsivity-like disinhibiting response. Excessive lever responses within 2 s after the initial response had no consequences and were recorded as cluster responses.
Response latency was defined as the time between the delivery of the S1 and the animal’s response on the lever. The average response latency for each test session was defined as the mean response latency of all trials for which a positive response occurred. Average response latency for each test session was defined as the mean response latency of all trials for which the animal responded.After training in this procedure, rats were tested daily for one month to allow them to reach and maintain a stable baseline reward threshold.
The ICSS experiments were carried out in the same cohort of rats in which effects of D-amphetamine administration were assessed.
Food intake and body weight measurements
Home cage food intake was measured at 2h, 6h, and 24h time points relative to -10 min D-amphetaminepretreatment. Pre-weighed food was providedat dark-cycle onset, 10minutes after drug administration.24h rat body weight change was calculated as the difference between the 24 h and the zero measurements.
Quantitative RT-PCR
Rats were deeply anesthetized with isoflurane and immediately decapitated. The extracted brain was immediately sliced using a rat brain matrix in 2 mm slices on an ice cold metal stage. Following coordinates in the Paxinos and Watson rat brain atlas, punches containing dorsal striatum (DS), nucleus accumbens (NAcc), ventromedial and lateral hypothalamus (VMH and LH), and ventral tegmental area (VTA) were obtained using a1 mm needle (for the VTA) and a 2 mm needle (for the rest of the areas) (Fine Science Tools, Inc., Foster City, CA)DS and the NAcc were not further dissected in their sub-regions and they were analyzed as a whole. Samples were quickly frozen at -80°C until further processing(Dore et al. 2013b). RNA extraction, retrotranscription, and theSYBR green-based real-time PCR were performed following previously published protocol.(Baiamonte et al. 2014; Cottone et al. 2012)The total RNA was extracted using the RNeasy lipid mini kit (Qiagen, Valencia, CA, UA), quantified using Nanodrop 1000 (Thermo Scientific, Wilmington, DE, USA) and reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). For quantitative real-time PCR, the Roche Light Cycler 480 Master-plus SYBR Green mix (Roche Applied Science, Indianapolis, IN, USA) was used. The reactions were carried out in a 10 μl volume using a 96-well plate Realplex2 machine (Eppendorf, Hamburg, Germany). Primers (0.5 μM final concentration, Sigma Aldrich), synthesized with a standard desalting purification, were the following: Cyclophilin A (Cyp), 5′-TAT CTG CAC TGC CAA GAC TGA GTG -3′ and 5′-CTT CTT GCT GGT CTT GCC ATT CC -3′; D1R, 5′- GAA GCA AAT CCG GCG CAT CTC -3′ and 5′-TTC AGA CTG GGC GCA TTC GAC-3′; D2R,5′- CCT TAA GAC GAT GAG CCG CAG AA -3′ and 5′-GGT TGA CGG CAC TGT TGA CAT AGC -3′; TH, 5′-GTC ACG CTG AAG GGC CTC TAT GCT -3′ and 5′- CTT CAA GAA GCG GGA CAC GTC CTC -3′. Cyp sequence was amplified using a three-temperature protocol which included an initial 10 min at 95 °C to activate Taq polymerase, followed by 40 denaturation cycles at 95 °C for 20 sec, then annealing at 58 °C for 15 sec, and extension at 72 °C for 10 sec. D1R, D2Rand TH sequences were amplified with a three-temperature protocol after an initial 10-min at 95 °C: 40 cycles at 95 °C for 15 sec, annealing at 59.3 °C for 10 sec and extension at 72 °C for 15 sec. The standard curves were constructed using purified fragments and the gene-specific amplification was determined by melting curve analysis with one peak at the expected melting temperature. The results were analyzed by the second derivative methods, expressed in arbitrary units,and normalized by the expression level of theCyp, the reference gene, in each brain area.
Fat pad and body composition analysis
DIO and DR rats, after prolonged feeding ad libitumwith chow diet or high-fat diet, were deeply anaesthetized with isoflurane and decapitated. Carcasses were weighed and bodies were transferred in dry ice to the University of Alabama–Birmingham where the fat pad and body composition analysis was carried out as previously described. (Cottone et al. 2013; Cottone et al. 2007) Briefly, carcasses were thawed at room temperature and weighed to determine freezing-related water loss. Gastrointestinal tracts were removed to determine eviscerated weight. Visceral (gonadal, retroperitoneal, mesenteric) and non-visceral (inguinal, subcutaneous) white fat pads and brown adipose tissue were dissected, weighed, and returned to the carcass for composition analysis. Total body water, fat mass, and fat-free dry mass (FFDM) were determined as in Harris and Martin (1984).
Statistical analysis
ICSS threshold,food intake and body weight change were analyzed using mixed two-way ANOVAs with Genotype as a between-subjects factor and Treatment as a within-subject factor. When a statistically significant overall effect and/or interaction were observed, data were further analyzed using separate one-way ANOVAs. The ICSS latency data were analyzed using the non-parametric Friedman analysis. Pairwise post-hoc comparisons were performed using the Student's t test (to compare two groups) or Fisher's least significant difference (LSD)analysis for all other comparisons, after confirmedsignificant omnibus effect (p≤0.05). Data obtained by the carcass analysis were analyzed using the Student’s t test to compare values from the two genotypes.
The statistical software used were Systat 11.0 (SPSS, Chicago, IL), Instat 3.0 (GraphPad, San Diego, CA), and Statistica 7.0 (StatSoft. Inc., Tulsa, OK). The graphical software used was SigmaPlot12.5 (SystatSofware Inc., Chicago, IL).
Supplemental Figure 1
Suppl Fig. 1. Dopamine 1 receptor (D1R) and dopamine 2 receptor (D2R) mRNA expression(A, C) in the lateral hypothalamus (LH) and (B, D) in the ventromedial hypothalamus (VMH) in (A, B)chow-fed and (C, D)high-fat diet fed DIO and DR rats (n=7-9/genotype). Data represent M±SEM expressed as percent of DR rats.
Supplemental References
Baiamonte BA, Valenza M, Roltsch EA, Whitaker AM, Baynes BB, Sabino V, Gilpin NW (2014) Nicotine dependence produces hyperalgesia: Role of corticotropin-releasing factor-1 receptors (CRF1Rs) in the central amygdala (CeA). Neuropharmacology 77: 217-23.
Cottone P, Sabino V, Nagy TR, Coscina DV, Levin BE, Zorrilla EP (2013) Centrally administered urocortin 2 decreases gorging on high-fat diet in both diet-induced obesity-prone and -resistant rats. Int J Obes (Lond) 37: 1515-23.
Cottone P, Sabino V, Nagy TR, Coscina DV, Zorrilla EP (2007) Feeding microstructure in diet-induced obesity susceptible versus resistant rats: central effects of urocortin 2. J Physiol 583: 487-504.
Cottone P, Wang X, Park JW, Valenza M, Blasio A, Kwak J, Iyer MR, Steardo L, Rice KC, Hayashi T, Sabino V (2012) Antagonism of Sigma-1 Receptors Blocks Compulsive-Like Eating. Neuropsychopharmacology.
Dore R, Iemolo A, Smith KL, Wang X, Cottone P, Sabino V (2013a) CRF mediates the anxiogenic and anti-rewarding, but not the anorectic effects of PACAP. Neuropsychopharmacology 38: 2160-9.
Dore R, Valenza M, Wang X, Rice KC, Sabino V, Cottone P (2013b) The inverse agonist of CB receptor SR141716 blocks compulsive eating of palatable food. Addict Biol.
Esposito R, Kornetsky C (1977) Morphine lowering of self-stimulation thresholds: lack of tolerance with long-term administration. Science 195: 189-91.
Esposito RU, Perry W, Kornetsky C (1980) Effects of d-amphetamine and naloxone on brain stimulation reward. Psychopharmacology (Berl) 69: 187-91.
Grilly DM, Loveland A (2001) What is a "low dose" of d-amphetamine for inducing behavioral effects in laboratory rats? Psychopharmacology (Berl) 153: 155-69.
Harris RB, Martin RJ (1984) Recovery of body weight from below "set point" in mature female rats. J Nutr 114: 1143-50.
Iemolo A, Valenza M, Tozier L, Knapp CM, Kornetsky C, Steardo L, Sabino V, Cottone P (2012) Withdrawal from chronic, intermittent access to a highly palatable food induces depressive-like behavior in compulsive eating rats. Behav Pharmacol 23: 593-602.
Levin BE, Dunn-Meynell AA, Balkan B, Keesey RE (1997) Selective breeding for diet-induced obesity and resistance in Sprague-Dawley rats. Am J Physiol 273: R725-30.
Marcus R, Kornetsk.C (1974) Negative and Positive Intracranial Reinforcement Thresholds - Effects of Morphine. Psychopharmacologia 38: 1-13.
Markou A, Koob GF (1991) Postcocaine anhedonia. An animal model of cocaine withdrawal. Neuropsychopharmacology 4: 17-26.
Pellegrino LJ, Pellegrino AS, Cushman AJ (1979) A stereotaxic atlas of the rat brain, 2d edn. Plenum Press, New York
Ricci MR, Levin BE (2003) Ontogeny of diet-induced obesity in selectively bred Sprague-Dawley rats. Am J Physiol Regul Integr Comp Physiol 285: R610-8.
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