Role of Hypothalamic Serotonin Receptors in Controlling Food Intake Following Pharmacological Administration of Leptin

Abstract

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...