Dopamine receptor stimulation regulates expression of developmental genes and disrupts netrin-1-mediated axon guidance

Abstract

Embryonic dopamine (DA) receptor (DR) expression in the medial frontal cortex (mFC) coincides with the formation of neural circuits during the period of axon pathfinding but the signaling properties of DRs and their effect on axon guidance events has not been examined. We used in situ hybridization and quantitative PCR to map DR mRNA transcripts in the medial frontal cortex (mFC) of the rat from E15 to E21. The developmental trajectory of DR mRNAs revealed distinct patterns of DA receptors 1 and 2 (DRD1, DRD2). To identify the functional properties of DRs during embryonic development, the phosphorylation states of CREB, ERK1/2, and GSK3β were examined after DR stimulation in primary neuronal cultures obtained from E15 and E18 embryos and cultured for three days to ensure a stable baseline level. DR-mediated signaling cascades were functional in E15 cultures. Because DA fibers do not reach the mFC by E15, and DA was not present in cultures, these data indicate that DRs can become functional in the absence of DA innervation and during axon pathfinding. We assessed the consequences of DR stimulation during Netrin-1-mediated axon guidance. Netrin-1 is a secreted axon guidance molecule expressed at the ventricular zone that can both attract or repel axons. We examined primary neuronal cultures of the mFC and found DRs co-localized with netrin-1 receptors DCC and UNC5C. In neuronal outgrowth assays as well as tissue explants, DR agonists interfered with the attraction of neurites to netrin-1. Stimulation of DRs in mFC cultures altered mRNA expression of UNC5C, suggesting that DR activation may modify the growth cone response to netrin-1 by regulating receptor abundance. By disrupting the trajectory of axon outgrowth, abnormal DR activity during embryogenesis may impact neuronal circuit formation in the forebrain and mFC function later in life.