P2Y1 receptor mediated neuronal fibre outgrowth in organotypic brain slice co-cultures. (June 2015)

• Record Type: Journal Article
• Authors: Heine, Claudia; Sygnecka, Katja; Scherf, Nico; Grohmann, Marcus; Bräsigk, Annett; Franke, Heike
• Is Part Of: Neuropharmacology. Volume 93(2015)
• Page Start: 252
• Page End: 266
• Publication Date: 2015-06
• Subjects: Purinergic stimulation -- P2Y1 receptors -- Organotypic slice co-culture -- Dopaminergic system -- Fiber growth -- PI3K/Akt -- ERK1/2; ACSF artificial cerebrospinal fluid -- ADPβS adenosine-5′-O-(2-thiodiphosphate) -- Akt serine–threonine kinase Akt -- ANOVA analysis of variance -- cLSM confocal laser scanning microscope -- DAB 3, 3′-diaminobenzidine hydrochloride -- DAergic dopaminergic -- DG dentate gyrus -- DIV days in vitro -- EC entorhinal cortex -- ERK extracellular signal regulated protein kinase -- FCS fetal calf serum -- GABA gamma-aminobutyric acid -- GFAP glial fibrillary acidic protein -- H hippocampus -- Hoe Hoechst -- HS horse serum -- IR immunoreactivity -- LDH lactate dehydrogenase -- MAP2 microtubule associated protein-2 -- MAPK mitogen-activated protein kinase -- MRS2500 (1R*, 2S*)-4-[2-Iodo-6-(methylamino)9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol -- NB-A Neurobasal-A -- NGF nerve growth factor -- P postnatal day -- P2Y1R−/− P2Y1R knockout mice -- PB phosphate buffer -- PFC prefrontal cortex -- PI3K phosphatidylinositide 3-kinase -- PPADS pyridoxal-phosphate-6-azophenyl-2′, 4′-disulphonic acid -- R receptor -- qPCR quantitative real-time polymerase chain reaction -- STR striatum -- TBS tris buffered saline -- TH tyrosine hydroxylase -- VTA/SN ventral tegmental area/substantia nigra -- WT wild-type; Neuropsychopharmacology -- Periodicals; Autonomic Agents -- Periodicals; Neuropsychopharmacologie -- Périodiques; Neuropsychopharmacology; Periodicals; Electronic journals; 615.78
• DOI: 10.1016/j.neuropharm.2015.02.001
• Languages: English
• ISSNs: 0028-3908

Abstract

Abstract: Extracellular purines have multiple functional roles in development, plastic remodelling, and regeneration of the CNS by stimulating certain P2X/Y receptor (R) subtypes. In the present study we elucidated the involvement of P2YRs in neuronal fibre outgrowth in the developing nervous system. We particularly focused on the P2Y1 R subtype and the dopaminergic system, respectively. For this purpose, we used organotypic slice co-cultures consisting of the ventral tegmental area/substantia nigra (VTA/SN) and the prefrontal cortex (PFC). After detecting the presence of the P2Y1 R in VTA/SN, PFC, and on outgrowing fibres in the border region (e.g. on glial processes) connecting both brain slices, we could show that pharmacological modulation of the receptor influenced neuronal fibre outgrowth. Biocytin-tracing and tyrosine hydroxylase-immunolabelling together with quantitative image analysis revealed a significant increase in fibre growth in the border region of the co-cultures after treatment with ADPβS (P2Y1, 12, 13 R agonist). The observed stimulatory potential of ADPβS was inhibited by pre-treatment with the P2X/YR antagonist PPADS. In P2Y1 R knockout (P2Y1 R −/− ) mice, the ADPβS-induced stimulatory effect was absent, while growth was significantly enhanced in the co-cultures of the respective wild-type. This observation was confirmed in entorhino-hippocampal co-cultures, an example of a different projection system, expressing the P2Y1 R. Using wortmannin and PD98059 we further showed that PI3K/Akt and MAPK/ERK cascades are involved in the mechanism underlying ADPβS-induced fibre growth. In conclusion, the data of this study clearly indicate that activation of the P2Y1 R stimulates fibre growth and thereby emphasises the general role of this particular receptor subtype during development and regeneration. Highlights: The P2Y1 R is expressed on neurons and astrocytes and co-localised with TH, GABA and glutamate within the mesocortical system. ADPβS stimulates neuronal fibre outgrowth in dopaminergic and hippocampal co-cultures. ADPβS has no growth promoting effect in P2Y1 R −/− mice co-cultures compared to the WT. ADPβS significantly enhances the outgrowth of TH-positive fibres originating in the VTA/SN. The PI3K/Akt and MAPK/ERK cascades are involved in ADPβS-promoted fibre growth.