CLINICAL STUDY Melanocortin 4 receptor distribution in the human hypothalamus Jacqueline E Siljee 1 , Unga A Unmehopa 2 , Andries Kalsbeek 1,3 , Dick F Swaab 2 , Eric Fliers 1 and Anneke Alkemade 1,4 1 Department of Endocrinology and Metabolism, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, Room G2-118, 1105 AZ Amsterdam, The Netherlands, Departments of 2 Neuropsychiatric Disorders and 3 Hypothalamic Integration Mechanisms, Netherlands Institute for Neuroscience, an Institute of the Royal Netherlands Academy of Arts and Science, 1105 BA Amsterdam, The Netherlands and 4 Alan Turing Institute, 1311 RL Almere, The Netherlands (Correspondence should be addressed to J E Siljee; Email: siljeej@gmail.com) Abstract Objective: The melanocortin 4 receptor (MC4R) is an essential regulator of energy homeostasis and metabolism, and MC4R mutations represent the most prevalent monogenetic cause of obesity in humans known to date. Hypothalamic MC4Rs in rodents are well characterized in neuroanatomical and functional terms, but their expression pattern in the human hypothalamus is unknown. Design and methods: To determine the topographic distribution and identity of cells expressing MC4R mRNA in the human hypothalamus, locked nucleic acid in situ hybridization was performed on nine human postmortem hypothalami. In addition, co-expression of MC4R with glial fibrillary acidic protein (GFAP), vasopressin/oxytocin (AVP/OXT), corticotropin-releasing hormone (CRH), neuropeptide Y (NPY), agouti-related protein (AgRP), and a-melanocyte stimulating hormone (a-MSH) was examined. Results: Most intense MC4R mRNA expression was present in the paraventricular nucleus (PVN), the supraoptic nucleus (SON), and the nucleus basalis of Meynert. Most MC4R-positive cells in the SON also expressed AVP/OXT. Co-expression with AVP/OXT in the PVN was less abundant. We did not observe co-expression of MC4R mRNA and GFAP, CRH, NPY, AgRP, or a-MSH. However, fiber-like stainingof NPY, AgRP, and a-MSH was found adjacent to MC4R-positive cells in the PVN. Conclusion: Expression of MC4R mRNA in the human hypothalamus is widespread and in close approximation to endogenous MC4R binding partners AgRP and a-MSH. European Journal of Endocrinology 168 361–369 Introduction The melanocortin 4 receptor (MC4R) plays an essential role in the maintenance of energy balance and is stimulated by endogenous melanocortins. MC4R has high affinity for a-melanocyte stimulating hormone (a-MSH) (1), while the receptor is inhibited by endo- genous agouti-related protein (AgRP) (2). Stimulation of MC4R decreases food intake and increases energy expenditure (3, 4). Less is known about the regulation of hypothalamic MC4R expression. The important role that MC4R plays in energy homeostasis is underlined by several observations: in rodents as well as in humans, heterozygous and homozygous mutations in MC4R lead to severe obesity. MC4R heterozygosity accounts for 2.5–6% of all cases of morbid childhood-onset obesity (5, 6, 7) and more than 90 obesity-associated MC4R mutations have been identified to date (8). In addition to obesity, mutations in one or both alleles of MC4R in humans are also associated with elevated fasting insulin levels, elevated blood glucose levels, enhanced linear growth, and incompletely suppressed GH secretion, as well as a reduction in blood pressure and heart rate, while a decreased prevalence of hypertension is also associated with mutations in MC4R (9, 10, 11, 12). To obtain more insight into the role of MC4R in hypothalamic melanocortin signaling, a better understanding of the underlying functional neuroanatomy is crucial. In spite of the severe metabolic phenotype and the high prevalence of MC4R abnormalities, no distribution studies that map MC4R expression in the human brain have been published. mRNA analysis in mice and rats (13, 14), as well as transgenic approaches (15), has shown that MC4R expression is restricted to the CNS and many functional experiments underline the importance of MC4R function in the hypothalamus (16, 17). Therefore, we set out to examine for the first time the expression of the MC4R in the human hypothalamus. We chose a locked nucleic acid (LNA) in situ hybridization approach, as reliable MC4R antisera for immunohistochemistry in human brain are not available. European Journal of Endocrinology (2013) 168 361–369 ISSN 0804-4643 q 2013 European Society of Endocrinology DOI: 10.1530/EJE-12-0750 Online version via www.eje-online.org