Intracellular Distribution of Melanin-concentrating Hormone in Hypothalamic Neurons : A Double Immunofluorescence Labelling Study

Energy homeostasis is defined by peripheral and central mechanisms, which together form a complex neuroendocrine network of processes ensuring the balance between food intake and energy expenditure. A key role has been implicated for the melanin-concentrating hormone, a cyclic, orexigenic peptide hormone produced in the zona incerta and the LHA. Besides food intake and energy expenditure, MCH is, amongst others involved in the regulation of sleep and wakefulness, mood and anxiety. Despite extensive research, several aspects regarding the function and localisation of MCH neurons are still incompletely understood. Details of the intracellular colocalization of MCH with other neuropeptides, as well as with additional components of functional importance is a prominent point still awaiting further clarification. The experimental part of my thesis is connected to a research project aimed at providing information on this issue. My work consisted of setting of the experimental conditions of double immunfluorescence labelling for the colocalisation of MCH with MAP2, a reliable dendritic marker. As both primary antibodies were raised in rabbit, it was necessary to apply a specific technique to keep clear of crossreactions inducing false positive results. The technique chosen is based on the stepwise visualisation of the two antigens, along with the use of labelled monovalent Fab for the visualization of the first primary antibody (http://www.jireurope.com/technical/fab-blok.asp). Several variations have been checked, including the order of the application of the two primary antibodies and several different combinations of the dilutions of both the primary and secondary antibodies. An optimal setup has been found which indicated for the first time colocalization of MAP2 and MCH in neuronal processes. This work provides the basis of further research on the intracellular distribution of MCH. Refining and generalizing of the observations is underway in order to achieve more complex identification of MCH containing neuronal processes