P2 receptor expression in the dopaminergic system of the rat brain during development

Abstract

Extracellular ATP facilitates the release of dopamine via P2 receptor activation in parts of the mesolimbic system. To characterize P2X/Y receptor subtypes in the developing dopaminergic system, their expression in organotypic slice co-cultures including the ventral tegmental area/substantia nigra (VTA/SN) complex and the prefrontal cortex (PFC) was studied in comparison to the receptor expression in 3–5 day-old and adult rats. Reverse transcriptase–polymerase chain reaction (RT-PCR) with specific primers for the P2X 1,2,3,4,6,7 and P2Y 1 receptors in the tissue extracts of organotypic co-cultures revealed the presence of the P2X and P2Y receptor mRNAs investigated. Multiple immunofluorescence labeling of the P2X/Y receptor protein indicated differences in the regional expression in the organotypic co-cultures after 10 days of cultivation (VTA/SN, P2X 1,2,3,4,6,7, P2Y 1,6,12; PFC, P2X 1,3,4,6,7, P2Y 1,2,4,6,12). At postnatal days 3–5, an immunofluorescence mostly comparable to that of adult rats was observed (VTA/SN and PFC: P2X 1,2,3,4,6,7, P2Y 1,2,4,6,12). There was one important exception: the P2X 7 receptor immunocytochemistry was not found in adult tissue, suggesting a potential role of this receptor in the development. Only few P2 receptors (e.g. P2X 1, P2Y 1) were expressed at fibers interconnecting the dopaminergic VTA/SN with the PFC in the organotypic co-cultures. The treatment of the cultures with the ATP analogues 2-methylthio-ATP and α,β-methylene-ATP induced an increase in axonal outgrowth and fiber density, which could be inhibited by pre-treatment with the P2X/Y receptor antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid. The co-localization of the dopamine-(D1) receptor with the P2X 1 receptor in organotypic slice cultures was evident. In the PFC of the co-cultures, and that of young but not adult rats, a number of tyrosine hydroxylase (TH)–positive cells also possessed P2Y 1-immunoreactivity (IR). Additionally, a strong P2Y 1-IR was observed on astrocytes. The present results show a time-, region- and cell type–dependent in vitro and in vivo expression pattern of different P2 receptor subtypes in the dopaminergic system indicating the involvement of ATP and its receptors in neuronal development and growth.