Obesity is prevalent in the United States. Diet-induced obesity (DIO) is the most common type of obesity and a major health-threatening chronic disease. Over-consumption of energy-dense, palatable food beyond basic caloric need promotes DIO. Recent data underpin a direct leptin function in the ventral tegmental area (VTA) of the midbrain reward circuitry to temper high-fat (HF) feeding. Our latest preliminary studies demonstrate (1) leptin activation of the VTA has a greater counter-regulatory function against HF intake and associated weight gain than that of the forebrain arcuate nucleus (ARC) of the hypothalamus; (2) a new physiological function of leptin to curtail weight gain in the substantia nigra (SN), another area within the midbrain reward circuitry. This proposal will investigate the role of leptin and leptin resistance specifically in the VTA and/or SN in modulating HF consumption and dietary weight gain and also address if these functions are especially important besides those in the hypothalamic ARC. We hypothesize that leptin function in the midbrain VTA, in particular, counters HF intake and contributes jointly with that in the forebrain ARC to the overall control of energy balance. We further postulate that HF feeding induces leptin resistance characterized by diminished food and body weight responses to leptin within the VTA, and that this resistance is a major culprit for exacerbating dietary obesity. Lastly, we propose a new role of leptin in energy homeostasis in the SN, and hypothesize that leptin directly regulates feeding and body weight in the SN and that leptin action in the SN contributes separately from that of the VTA to curb dietary weight gain. Three Specific Aims will be carried out in male Sprague Dawley (SD) rats. Aim 1 will address if leptin action in the VTA and in the ARC works in concert to curtail HF consumption and deter dietary weight gain. Aim 1 will also evaluate leptin function in the VTA to decrease the preference for HF food using a diet-selection paradigm compared to leptin effect in the ARC. Leptin will be chronically over-expressed via site-directed, recombinant adeno-associated virus-mediated (rAAV) gene delivery specifically to either the VTA, or ARC or both sites. Aim 2 seeks to identify a presumed leptin resistance in the VTA with chronic HF exposure and to establish a causal role of leptin resistance in the VTA to promote greater DIO. The onset of leptin resistance with chronic HF exposure in the VTA will be examined by assessing acute maximal leptin signaling in brain micro-punches and central leptin responsiveness. The latter will be determined from 24-hour food intake and body weight responses to a single dose of leptin introduced via implanted brain cannula in the VTA or ARC. Moreover, leptin resistance will be simulated by leptin receptor blockade achieved via rAAV-mediated leptin antagonist over-expression specifically in either the VTA or ARC, and the effect of this blockade in either site on HF consumption and dietary weight gain compared. Aim 3 is designed to validate the newly proposed leptin physiological function in the SN on energy homeostasis and/or reward eating. Potential effects of leptin in the SN will be compared to those in the VTA. Leptin will be over-expressed in the SN or VTA, or in both sites simultaneously via rAAV-leptin gene delivery in chow-raised rats remaining on chow or introduced to a HF diet. Food consumption, body weight gain, and energy expenditure including locomotor activity will be examined along with JAK2-STAT3 and/or ERK leptin signaling. It is of particular interest to explore if leptin over- expression in both the VTA and SN, simultaneously, will display an additive or synergistic inhibition of HF food intake and associated obesity compared to overexpression in either site alone. The outcomes of this application will provide valuable insights into new brain mechanisms governing reward eating and diet-induced obesity. The new information may lead to better strategies to address the looming obesity epidemic.