Arachidonic acid as a retrograde signal controlling growth and dynamics of retinotectal arbors

Abstract

In the developing visual system, correlated presynaptic activity between neighboring retinal ganglion cells (RGC) stabilizes retinotopic synapses via a postsynaptic NMDAR (N-methyl-D-aspartate receptor)-dependent mechanism. Blocking NMDARs makes individual axonal arbors larger, which underlies an unsharpened map, and also increases branch turnover, as if a stabilizing factor from the postsynaptic partner is no longer released. Arachidonic acid (AA), a candidate retrograde stabilizing factor, is released by cytoplasmic phospholipase A2 (cPLA2) after Ca(2+) entry through activated NMDARs, and can activate presynaptic protein kinase C to phosphorylate various substrates such as GAP43 to regulate cytoskeletal dynamics. To test the role of cPLA2 in the retinotectal system of developing zebrafish, we first used PED6, a fluorescent reporter of cPLA2 activity, to show that 1-3 min of strobe flashes activated tectal cPLA2 by an NMDAR-dependent mechanism. Second, we imaged the dynamic growth of retinal arbors during both local inhibition of tectal cPLA2 by a pharmacological inhibitor, arachidonic tri-fluoromethylketone, and its suppression by antisense oligonucleotides (both injected intraventricularly). Both methods produced larger arbors and faster branch dynamics as occurs with blocking NMDARs. In contrast, intraocular suppression of retinal cPLA2 with large doses of antisense oligos produced none of the effects of tectal cPLA2 inhibition. Finally, if AA is the retrograde messenger, the application of exogenous AA to the tectum should reverse the increased branch turnover caused by blocking either NMDARs or cPLA2. In both cases, intraventricular injection of AA stabilized the overall branch dynamics, bringing rates down below the normal values. The results suggest that AA generated postsynaptically by cPLA2 downstream of Ca(2+) entry through NMDARs acts as a retrograde signal to regulate the dynamic growth of retinal arbors.