Prolonged High Fat Diet Reduces Dopamine Reuptake without Altering DAT Gene Expression Jackson J. Cone 1 , Elena H. Chartoff 2 , David N. Potter 2 , Stephanie R. Ebner 3 , Mitchell F. Roitman 3 * 1 Graduate Program in Neuroscience, University of Illinois at Chicago, Chicago, Illinois, United States of America, 2 Dept. of Psychiatry, Harvard Medical School, McLean Hospital, Belmont, Massachusetts, United States of America, 3 Department of Psychology, University of Illinois at Chicago, Chicago, Illinois, United States of America Abstract The development of diet-induced obesity (DIO) can potently alter multiple aspects of dopamine signaling, including dopamine transporter (DAT) expression and dopamine reuptake. However, the time-course of diet-induced changes in DAT expression and function and whether such changes are dependent upon the development of DIO remains unresolved. Here, we fed rats a high (HFD) or low (LFD) fat diet for 2 or 6 weeks. Following diet exposure, rats were anesthetized with urethane and striatal DAT function was assessed by electrically stimulating the dopamine cell bodies in the ventral tegmental area (VTA) and recording resultant changes in dopamine concentration in the ventral striatum using fast-scan cyclic voltammetry. We also quantified the effect of HFD on membrane associated DAT in striatal cell fractions from a separate group of rats following exposure to the same diet protocol. Notably, none of our treatment groups differed in body weight. We found a deficit in the rate of dopamine reuptake in HFD rats relative to LFD rats after 6 but not 2 weeks of diet exposure. Additionally, the increase in evoked dopamine following a pharmacological challenge of cocaine was significantly attenuated in HFD relative to LFD rats. Western blot analysis revealed that there was no effect of diet on total DAT protein. However, 6 weeks of HFD exposure significantly reduced the 50 kDa DAT isoform in a synaptosomal membrane-associated fraction, but not in a fraction associated with recycling endosomes. Our data provide further evidence for diet-induced alterations in dopamine reuptake independent of changes in DAT production and demonstrates that such changes can manifest without the development of DIO. Citation: Cone JJ, Chartoff EH, Potter DN, Ebner SR, Roitman MF (2013) Prolonged High Fat Diet Reduces Dopamine Reuptake without Altering DAT Gene Expression. PLoS ONE 8(3): e58251. doi:10.1371/journal.pone.0058251 Editor: Sidney Arthur Simon, Duke University Medical Center, United States of America Received October 26, 2012; Accepted February 5, 2013; Published March 13, 2013 Copyright: ß 2013 Cone et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The project described was supported by National Institutes of Health (NIH) grants DA025634 (MFR)and T32-MH067631 from the Biomedical Neuroscience Training Program (JJC). Additional support was provided by the National Center for Research Resources and the National Center for Advancing Translational Sciences, NIH, through grant UL1RR029877 (JJC) and by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust (JJC). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the Chicago Biomedical Consortium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mroitman@uic.edu Introduction The overweight and obese represent an increasingly larger percentage of the United States and worldwide populations [1,2]. While there are many pathways to obesity, perhaps one of the biggest threats to healthy body weight is the prevalence and consumption of highly palatable, densely caloric foods [3]. Indeed, the energy density (kcal/g) of food potently contributes to overweight and obesity in adults [4,5]. Palatable foods evoke dopamine release in the striatum of both humans and non-human animals [6,7,8,9] and subjective ratings of fattiness in food are positively correlated with the strength of neural responses in the ventral striatum [10]. Thus, dopamine and the striatum appear to contribute to preferences for energy dense foods. Recently, it was shown that differences in diet can cause simultaneous changes in striatal circuitry and food-directed behavior [11]. However, perhaps less appreciated is the growing evidence that differences in ingested foods, especially with respect to fat, can feedback on and alter striatal dopamine signaling. Striatal dopamine signaling is regulated by several factors including dopamine production by the enzyme tyrosine hydrox- ylase, pre- and postsynaptic dopamine receptors, and presynaptic dopamine transporters (DATs), all of which have been implicated in obesity [12,13]. Alterations in DAT number or function can alter the sphere of influence of released dopamine and conse- quently striatal function [14,15]. Insulin, released in response to ingested food, has been shown to influence DAT function [16,17]. Thus, the DAT is one of the likely candidates for the effects of diet. Recently, correlations between obesity and DAT availability as well as diet-induced alterations of DAT function have been explored. Body mass index (BMI) is negatively correlated with DAT availability in the human striatum [18]. DAT binding, and hence availability, is reduced in high fat diet (HFD) fed mice [19]. HFD -induced obesity (DIO) is associated with a reduced rate of dopamine reuptake by the DAT in rats [20]. Taken together, these studies suggest that obesity established by HFD consumption can potently influence critical presynaptic regulators of dopamine signaling – especially the DAT. However, the time course of diet- induced alterations in dopamine signaling and whether the development of DIO is requisite for changes to manifest remains unknown. We assayed DAT function by evoking dopamine release in the ventral striatum and quantifying its rate of reuptake in rats using fast-scan cyclic voltammetry. To determine if decreased dopamine reuptake was caused by reduced DAT gene expression, we measured DAT mRNA in the ventral tegmental area and PLOS ONE | www.plosone.org 1 March 2013 | Volume 8 | Issue 3 | e58251