Validation of antibodies for neuroanatomical localization of the P2Y receptor in macaque brain.

Focus on the purinergic receptor P2Y has increased following the finding of an association between the sleep disorder narcolepsy and a genetic variant in P2RY11 causing decreased gene expression. Narcolepsy is believed to arise from an autoimmune destruction of the hypothalamic neurons that produce the neuropeptide hypocretin/orexin. It is unknown how a decrease in expression of P2Y might contribute to an autoimmune reaction towards the hypocretin neurons and the development of narcolepsy. To advance narcolepsy research it is therefore extremely important to determine the neuroanatomical localization of P2Y in the brain with particular emphasis on the hypocretin neurons. In this article we used western blot, staining of blood smears, and flow cytometry to select two antibodies for immunohistochemical staining of macaque monkey brain. Staining was seen in neuron-like structures in cortical and hypothalamic regions. Rats do not have a gene orthologue to the P2Y receptor and therefore rat brain was used as negative control tissue. The chromogenic signal observed in macaque monkey brain in neurons was not considered reliable, because the antibodies stained rat brain in a similar distribution pattern. Hence, the neuroanatomical localization of the P2Y receptor remains undetermined due to the lack of specific P2Y antibodies for brain immunohistochemistry.