No abstract

The onset and exacerbation of obesity involves the overproduction of the adipocyte-derived hormone leptin, a key mediator of homeostatic appetite regulation and a signal for satiety. Although leptin’s hypothalamic regulation of food intake has been extensively investigated, its role in tandem with the anorectic neurotransmitter serotonin (5-HT) has been less characterized. 5-HT is synthesized in the dorsal raphe nucleus (DRN) where anatomical projections to many hypothalamic nuclei have previously been identified. Preliminary studies in our lab have: (1) identified serotonergic neurons responsive to leptin in the DRN that project to the arcuate nucleus (ARC) of the hypothalamus and (2) demonstrated leptin injected into the DRN significantly decreases food intake. The objective of the current study was to identify the role of 5-HT in leptin’s regulation of food intake first within the DRN, then between the DRN and the ARC. Adult male Sprague Dawley rats underwent stereotaxic surgery for guide cannula implantation in the DRN. After recovery, animals were administered 100 µg of p-chlorophenylalanine (PCPA), an inhibitor of 5-HT synthesis, in the DRN each day for four days. On the fourth day, leptin was also administered in the DRN (5 µg/rat) and food intake was measured over a 24-hour time course. ANOVA analysis revealed a significant difference in 24-hour food intake [F (3, 18) = 3.972; P = 0.0246] and post-hoc analysis showed that animals treated with leptin significantly decreased food intake (17.2 ± 2.0 g) compared to control rats (25.4 ± 0.9 g), whereas PCPA-treated rats did not differ from the control rats, suggesting that depletion of 5-HT attenuated leptin’s ability to regulate food intake within the DRN. To examine the role of 5-HT on leptin’s hypothalamic action, a subsequent experiment was conducted by implanting an additional cannula into the ARC for the administration of leptin or vehicle on the fourth day of treatment. ANOVA analysis revealed a significant difference in 24-hour food intake [F (3, 16) = 5.998; P = 0.0061] and post-hoc analysis showed that only rats treated with leptin in the ARC significantly decreased food intake (14.0 ± 1.5 g) compared to controls (21.8 ± 0.5 g). 5-HT depletion was assessed post-mortem using immunohistochemistry and was later quantified. Collectively, these results demonstrate that leptin’s ability to regulate food intake is dependent on 5-HT, regardless of the area of regulation (i.e. DRN or the hypothalamus).

Leptin is a homeostatic regulatory element that signals the presence of energy stores -in the form of adipocytes- which ultimately reduces food intake and increases energy expenditure. Similarly, serotonin (5-HT), a signaling molecule found in both the central and peripheral nervous systems, also regulates food intake. Here we use a combination of pharmacological manipulations, optogenetics, retrograde tracing, and in situ hybridization, combined with behavioral endpoints to physiologically and anatomically identify a novel leptin-mediated pathway between 5-HT neurons in the dorsal raphe nucleus (DRN) and hypothalamic arcuate nucleus (ARC) that controls food intake. In this study, we show that microinjecting leptin directly into the DRN reduces food intake in male Sprague-Dawley rats. This effect is mediated by leptin-receptor expressing neurons in the DRN as selective optogenetic activation of these neurons at either their ARC terminals or DRN cell bodies also reduces food intake. Anatomically, we identified a unique population of serotonergic raphe neurons expressing leptin receptors that send projections to the ARC. Finally, by utilizing in vivo microdialysis and high-performance liquid chromatography, we show that leptin administration to the DRN increases 5-HT efflux into the ARC. Overall, this study identifies a novel circuit for leptin-mediated control of food intake through a DRN-ARC pathway, utilizing 5-HT as a mechanism to control feeding behavior. Characterization of this new pathway creates opportunities for understanding how the brain controls eating behavior, as well as opens alternative routes for the treatment of eating disorders.

Use of leptin as a pharmacological intervention for obesity has failed since its discovery in 1994. This failure is attributed to the already high levels of leptin circulating in most obese patients, contributing to the lack of response from the brain, termed “leptin resistance”. Still there is hope that leptin may yet be used as a treatment for obesity. Leptin receptors are expressed throughout the brain, showing that the anorectic function of leptin does not rely solely on direct stimulation of hypothalamic neurons. In our laboratory, using pharmacological and optogenetic approaches we have demonstrated that food intake is also controlled by the leptin receptors located in the dorsal raphe nuclei (DRN). We have also utilized retrograde tracing to characterize a neural pathway from the DRN to the arcuate of the hypothalamus that is dependent on leptin. Then, through pharmacological suppression of serotonin (5HT) production, we showed that an intact pool of 5HT in the DRN is necessary for leptin to reduce food intake. Therefore, our objective for this study is to understand the role of specific serotonin receptors in the anorectic effects of leptin through the raphe. We hypothesize that both the 2C and 1B 5HT receptors are necessary for the anorectic effects of leptin through the raphe, and only blocking one will show partial decrease of leptin’s effects. To address this hypothesis, we performed stereotaxic surgery on adult male Sprague Dawley rats to implant cannulas into the third ventricle in the vicinity of hypothalamic nuclei. Following recovery, they were habituated to metabolic cages for 7 days. Then, we administered selective antagonists for the 5HT2C (SB242084) and/or 5HT1B (GR55562) receptors (or vehicle as control) into the third ventricle to block these receptors throughout the hypothalamus. Thirty minutes later, we administered leptin to the DRN, done just before the onset of the dark cycle. For 24 hours, the metabolic cages automatically measured food intake. Five days after the administration, we reversed groups, giving vehicle to the antagonist treated groups and vice versa, recording food intake in the same way. Five days after the second administration, we injected antagonist or vehicle into the third ventricle, then leptin into the DRN 30 minutes after. Two hours post leptin injection, rats were perfused and fixed with 4% paraformaldehyde and brains were collected. We then sectioned and performed immunofluorescence to label cFOS, a marker of neuron activation, in the hypothalamus. CFos was counted to determine differences between animals treated with antagonist vs vehicle. We observed that food intake was reduced by leptin just as we’ve shown previously. However, this anorectic effect is lessened when the 5HT1B or 5HT2C receptor is given into the third ventricle prior to leptin infusion, but not completely removed. These results strongly suggest that the anorectic effects of leptin and serotonin are strongly intertwined, and understanding this interaction may lead to better pharmacological trea...